WO2023234416A1 - Substance d'affinité, composé, anticorps et leurs sels - Google Patents

Substance d'affinité, composé, anticorps et leurs sels Download PDF

Info

Publication number
WO2023234416A1
WO2023234416A1 PCT/JP2023/020712 JP2023020712W WO2023234416A1 WO 2023234416 A1 WO2023234416 A1 WO 2023234416A1 JP 2023020712 W JP2023020712 W JP 2023020712W WO 2023234416 A1 WO2023234416 A1 WO 2023234416A1
Authority
WO
WIPO (PCT)
Prior art keywords
affinity
salt
antibody
substance
group
Prior art date
Application number
PCT/JP2023/020712
Other languages
English (en)
Japanese (ja)
Inventor
豊 松田
友博 藤井
健一郎 伊藤
一敏 ▲高▼橋
吉彦 松田
浩輝 山口
直子 津吉
隼人 長野
梨花 ▲高▼杉
Original Assignee
味の素株式会社
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 味の素株式会社 filed Critical 味の素株式会社
Publication of WO2023234416A1 publication Critical patent/WO2023234416A1/fr

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/55—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present invention relates to affinity substances and salts thereof, as well as compounds containing affinity substances, antibodies, and salts thereof.
  • ADC Antibody Drug Conjugates
  • ADC is a drug consisting of an antibody conjugated with a drug (eg, an anticancer drug), and has direct cell-killing activity against cancer cells and the like.
  • a typical ADC is T-DM1 (trade name: Kadcyla (registered trademark)), which was jointly developed by Immunogene and Roche.
  • Nonuniformity has been a problem with ADCs such as T-DM1 since the beginning of their development.
  • DAR drug-antibody ratio
  • conjugation position are not constant. It is known that such a random conjugation method usually results in a DAR in the range of 0 to 8, resulting in the production of multiple antibody drugs with different numbers of drug bonds.
  • changing the number and bonding position of drugs in ADCs changes the pharmacokinetics, drug release rate, and effects. For these reasons, next-generation ADCs are required to control the number and position of conjugated drugs. It is believed that if the number and position are constant, the problems of expected efficiency, variations in conjugated drugs, and lot differences, so-called regulation, will be solved.
  • C-CAP Chemical Conjugation by Affinity Peptide
  • This method has succeeded in regioselectively modifying antibodies by reacting with antibodies a peptide reagent in which an NHS activated ester and a drug are linked to affinity peptides.
  • the antibody and the drug are linked via a linker containing a peptide moiety.
  • Peptide moieties have potential immunogenicity and are also susceptible to hydrolysis in blood. Therefore, the ADC produced by this method has room for improvement in that it includes a peptide moiety in the linker.
  • an antibody that does not contain a peptide moiety as a linker and has a functional substance (e.g., a drug) in a regioselective manner is produced by a chemical synthesis method using a predetermined compound containing an affinity peptide.
  • Techniques for preparation have been reported (Patent Documents 2 to 5).
  • Patent Document 6 describes a method for easily producing a large amount of affinity peptides by using an affinity peptide containing glutamine-glutamic acid-threonine (QET) at the N-terminus in the production of compounds containing affinity peptides. The technology has been disclosed. Avoiding the use of linkers containing peptide moieties is desirable in clinical applications.
  • Patent Document 6 describes (i) various receptors (e.g., fibroblast growth factor receptor (FGFR), hepatocyte growth factor receptor (HGFR/c-Met), erythropoietin receptor, thrombopoietin receptor); ), etc., and a peptide molecule containing a peptide linker can modulate the activity of the protein by non-covalently binding to the protein, and (ii) It is disclosed that a peptide linker of a predetermined length can be used that includes an amino acid sequence consisting of the amino acid residues proline (P), alanine (A), and serine (S).
  • FGFR fibroblast growth factor receptor
  • HGFR/c-Met hepatocyte growth factor receptor
  • S serine
  • An object of the present invention is to develop a technology that can easily chemically modify only one heavy chain in an antibody constitutional unit (in other words, an immunoglobulin unit containing two heavy chains and, if necessary, two light chains). It is to develop.
  • a further object of the present invention is to develop antibodies that are regioselectively modified while easily chemically modifying only one heavy chain in the antibody structural unit.
  • an affinity substance comprising a first and second affinity portion having affinity for the constant region of an antibody heavy chain, and (B) a reactive group for antibodies. It has been found that by using a compound containing or a salt thereof, only one heavy chain in the structural unit of an antibody can be easily chemically modified.
  • the compound of the present invention or a salt thereof can bind to the two constituent units of an antibody through an affinity substance (A) comprising first and second affinity parts that have affinity for the constant region of the antibody heavy chain. can be associated with a heavy chain and then react specifically with the side chain of a particular amino acid residue in one heavy chain in the antibody structural unit through the antibody-reactive group (R). , it is possible to produce an affinity substance-modified antibody or a salt thereof in which only one of the two heavy chains in the antibody structural unit is modified (FIG. 1).
  • the mechanism of modification of only one heavy chain in the structural unit of an antibody by the compound of the present invention or a salt thereof is as follows.
  • the affinity substance (including the first and second affinity parts having affinity for the constant region in the heavy chain of an antibody) contained in the compound of the present invention or a salt thereof is a substance that has an affinity for the constant region of the antibody heavy chain.
  • the reactive group contained in the compound of the present invention or its salt can modify only one heavy chain ( Figure 1).
  • the constant region of the other heavy chain (the constant region of the unmodified heavy chain) is associated with an affinity moiety and is sterically hindered, so that the other molecule (the compound of the present invention or its salt) cannot , cannot associate with the constant region of the other heavy chain via the affinity substance it contains.
  • the compound of the present invention or a salt thereof can associate with the constant regions of two heavy chains via two affinity moieties contained in the affinity substance, and therefore can stably associate with the constituent units of antibodies. Therefore, the association of other molecules with the antibody constituent units can be highly inhibited. Therefore, the compound of the present invention or a salt thereof can highly suppress modification of the constant region of the other heavy chain, and therefore can modify only the constant region of one heavy chain (FIG. 1).
  • Patent Documents 1 to 5 disclose that an antibody can be regioselectively modified with a functional substance by using an affinity substance and a compound containing a reactive group for the antibody. and (2) to develop a technology that can easily chemically modify only one heavy chain of an antibody.
  • the technical concept of chemically modifying only one heavy chain in the constitutional unit of an antibody by using a substance that has an affinity for the antibody and a compound or its salt that contains a reactive group for the antibody I haven't even suggested it.
  • Patent Document 6 discloses that a peptide molecule containing the above-mentioned first and second physiologically active peptide sites for a protein and a predetermined peptide linker binds to the protein non-covalently, thereby increasing the activity of the protein.
  • the technical concept of chemically modifying only one heavy chain in a unit is neither described nor suggested.
  • an immunoglobulin unit comprising two heavy chains and optionally two light chains
  • Such antibodies include (a) an immunoglobulin unit comprising two heavy chains and optionally two light chains, and (b) a modifying unit (e.g., an affinity substance, a bioorthogonal functional group, (c) the modification unit is introduced only into the constant region of one of the heavy chains in the immunoglobulin unit.
  • a modifying unit e.g., an affinity substance, a bioorthogonal functional group
  • the present invention is as follows. [1] (A) an affinity substance comprising first and second affinity parts having affinity for the constant region in the heavy chain of an antibody, and (B) a compound or a salt thereof comprising a group reactive with the antibody. [2] The compound of [1] or a salt thereof, wherein the constant region is an Fc region. [3] The compound of [1] or [2] or a salt thereof, wherein the constant region is a CH2 domain. [4] The compound of any one of [1] to [3] or a salt thereof, wherein the constant region is a human constant region. [5] The compound of any one of [1] to [4] or a salt thereof, wherein the antibody is IgG.
  • the affinity substance has the following formula (A): AP1- LA -AP2 (A) [During the ceremony, AP1 indicates a first affinity peptide with affinity for the constant region in the heavy chain of antibodies; AP2 represents a second affinity peptide with affinity for the constant region in the heavy chain of antibodies; LA indicates a linker. ] The compound of any one of [1] to [6] or a salt thereof. [8] The affinity substance (i) (i-1) contains only one specific reactive group, and (i-2) is linked to a reactive group for the antibody via the specific reactive group.
  • the compound of any one of [1] to [7] or a salt thereof [9] The compound according to any one of [1] to [8], wherein the affinity substance is an affinity polypeptide comprising first and second affinity peptides that have affinity for the constant region in the heavy chain of an antibody. Or its salt.
  • the affinity polypeptide has the following formula (A'): AP1-PL A -AP2 (A') [During the ceremony, AP1 indicates a first affinity peptide that has affinity for the constant region in the heavy chain of an antibody and is present on the N-terminal side of the affinity polypeptide; AP2 indicates a second affinity peptide that has affinity for the constant region in the heavy chain of an antibody and is present on the C-terminal side of the affinity polypeptide; PLA indicates a peptide linker. ] The compound of any one of [1] to [6] or a salt thereof.
  • the affinity polypeptide (i) (i-1) contains only one amino acid residue having an amino group in its side chain, and (ii-2) has reactivity with the antibody through the amino group. or (ii) a reactive group for the antibody via the N-terminal amino group in the first affinity peptide. Or its salt.
  • the compound of any one of [1] to [12] or a salt thereof, wherein the affinity polypeptide further contains a tripeptide consisting of Gln-Glu-Thr (QET) at its N-terminus.
  • One of the first and second affinity peptides is an affinity peptide that has an affinity for a constant region in the heavy chain of an antibody and has one lysine residue, and The compound according to any one of [1] to [14] or a salt thereof, wherein the other of the affinity peptides is an affinity peptide that has an affinity for the constant region in the heavy chain of an antibody and does not have a lysine residue.
  • Affinity peptides that have affinity for the constant region in the heavy chain of antibodies and have one lysine residue are the following (1) to (4): (1) Affinity peptide comprising the amino acid sequence (Fc3K) of RGNCAYHKGQIIWCTYH (SEQ ID NO: 38); (2) In the amino acid sequence of RGNCAYHKGQIIWCTYH (SEQ ID NO: 38), one or two amino acid residues other than lysine residues and cysteine residues are replaced with other amino acid residues other than lysine residues and cysteine residues.
  • an affinity peptide comprising the amino acid sequence as described above and having affinity for the constant region in the heavy chain of an antibody; (3) an affinity peptide containing the amino acid sequence (Z34CK) of FNKQCQRRFYEALHDPNLNEEQRNARIRSIREEC (SEQ ID NO: 39); and (4) an affinity peptide containing the amino acid sequence (Z34CK) of FNKQCQRRFYEALHDPNLNEEQRNARIRSIREEC (SEQ ID NO: 39) other than lysine and cysteine residues.
  • pcs or 2 pieces an affinity peptide comprising an amino acid sequence in which the amino acid residue of is substituted with another amino acid residue other than a lysine residue or a cysteine residue, and having affinity for the constant region in the heavy chain of an antibody; (wherein the two cysteine residues included in the amino acid sequence may be cross-linked by a disulfide bond) and/or have an affinity for the constant region in the heavy chain of the antibody.
  • the affinity peptides having the following (5) to (10): (5) an affinity peptide comprising the amino acid sequence (Z34CM) of FNMQCQRRFYEALHDPNLNEEQRNARIRSIREEC (SEQ ID NO: 40); (6) Amino acids in which one or two amino acid residues other than cysteine residues in the amino acid sequence of FNMQCQRRFYEALHDPNLNEEQRNARIRSIREEC (SEQ ID NO: 40) are substituted with lysine residues and other amino acid residues other than cysteine residues.
  • an affinity peptide comprising a sequence and having an affinity for a constant region in an antibody heavy chain; (7) an affinity peptide comprising the amino acid sequence (ProAR) of FNREQQNAFYEILHLPNLNEEQRNGFIQSLRDDPSQSANLLAEA (SEQ ID NO: 41); (8) Amino acids in which one or two amino acid residues other than cysteine residues in the amino acid sequence of FNREQQNAFYEILHLPNLNEEQRNGFIQSLRDDPSQSANLLAEA (SEQ ID NO: 41) are substituted with lysine residues and other amino acid residues other than cysteine residues.
  • an affinity peptide comprising a sequence and having an affinity for a constant region in an antibody heavy chain
  • An affinity peptide comprising the amino acid sequence of RGNCAYHRGQIIWCTYH (SEQ ID NO: 78); and (10) In the amino acid sequence of RGNCAYHRGQIIWCTYH (SEQ ID NO: 78), one or two amino acid residues other than cysteine residues are lysine an affinity peptide comprising an amino acid sequence substituted with another amino acid residue other than a cysteine residue and a cysteine residue, and having an affinity for the constant region in the heavy chain of an antibody; (wherein the two cysteine residues included in the amino acid sequence may be cross-linked by a disulfide bond), the compound of [15] or a salt thereof.
  • the compound has the following formula (I): [During the ceremony, R represents the reactive group, L represents a linker; A indicates the affinity substance. ] The compound of any one of [1] to [16] or a salt thereof. [18] The compound according to any one of [1] to [17], wherein the compound or a salt thereof further includes (iii) a cleavable moiety between (i) the affinity substance and (ii) the reactive group. Or its salt. [19] The compound of [18] or a salt thereof, wherein the cleavable moiety is a cleavable moiety that can generate a bioorthogonal functional group on the reactive group side upon cleavage.
  • the compound has the following formula (Ia): [During the ceremony, R represents the reactive group, L 1 indicates the first linker, L2 represents a second linker; CLE (B) indicates a cleavable moiety that can generate a bioorthogonal functional group on the reactive group side by cleavage, A indicates the affinity substance. ] The compound of [19] or a salt thereof. [21] The compound has the following formula (Ia-1): [During the ceremony, X represents a leaving group, W 1 , W 2 and W 3 each independently represent an oxygen atom or a sulfur atom, L 3 indicates a third linker, L 4 indicates the fourth linker, S represents a sulfur atom, A indicates the affinity substance.
  • the compound has the following formula (Ib-1): [During the ceremony, X represents a leaving group, W 1 , W 2 and W 3 each independently represent an oxygen atom or a sulfur atom, L 7 indicates the seventh linker, L8 indicates the eighth linker, B represents a group containing a bioorthogonal functional group, V represents an oxygen atom or a sulfur atom, A indicates the affinity substance. ] The compound of [23] or a salt thereof.
  • the bioorthogonal functional group is an azide residue, an alkyne residue, a tetrazine residue, an alkene residue, a thiol residue, a maleimide residue, a thiol residue, a furan residue, or a halocarbonyl residue, Any compound of [23] to [25] or a salt thereof.
  • a reagent for antibody derivatization comprising the compound of any one of [1] to [26] or a salt thereof.
  • An affinity substance-modified antibody or a salt thereof containing an affinity substance in the constant region of the antibody heavy chain which contains an affinity substance containing first and second affinity parts having affinity for the constant region of the antibody heavy chain .
  • the affinity substance binds to the constant region of the one heavy chain through modification of the amino group in the side chain of a lysine residue present at one or more positions in the constant region of the one heavy chain.
  • the affinity substance-modified antibody has the following formula (II): [During the ceremony, Ig refers to an immunoglobulin unit containing two heavy chains and optionally two light chains; L represents a linker; A indicates the affinity substance, The average modification percentage r of the immunoglobulin units by the affinity substance is 65-135%.
  • affinity substance-modified antibody or its salt [34] The affinity substance-modified antibody or a salt thereof [33], wherein the cleavable moiety is a cleavable moiety that can generate a bioorthogonal functional group on the immunoglobulin unit side upon cleavage.
  • the affinity substance-modified antibody has the following formula (IIa): [During the ceremony, Ig indicates the immunoglobulin unit, L 1 indicates the first linker, L2 represents a second linker; CLE (B) represents a cleavable moiety that can generate a bioorthogonal functional group on the immunoglobulin unit side by cleavage; A indicates the affinity substance, The average modification percentage r of the immunoglobulin units by the affinity substance is 65-135%. ] The affinity substance-modified antibody of [34] or a salt thereof, comprising a structural unit represented by [34].
  • the affinity substance-modified antibody has the following formula (IIa-1): [During the ceremony, Ig indicates the immunoglobulin unit, W 1 , W 2 and W 3 each independently represent an oxygen atom or a sulfur atom, L 3 indicates a third linker, L 4 indicates the fourth linker, S represents a sulfur atom, A indicates the affinity substance, The average modification percentage r of the immunoglobulin units by the affinity substance is 65-135%. ] The affinity substance-modified antibody of [34] or a salt thereof, comprising a structural unit represented by [34].
  • the affinity substance-modified antibody has the following formula (IIb): [During the ceremony, Ig indicates the immunoglobulin unit, L 5 indicates the fifth linker, L 6 indicates the sixth linker, B represents a group containing a bioorthogonal functional group, CLE indicates a cleavable moiety; A indicates the affinity substance, The average modification percentage r of the immunoglobulin units by the affinity substance is 65-135%.
  • the affinity substance-modified antibody has the following formula (IIb-1): [During the ceremony, Ig indicates the immunoglobulin unit, W 1 , W 2 and W 3 each independently represent an oxygen atom or a sulfur atom, L 7 indicates the seventh linker, L8 indicates the eighth linker, B represents a group containing a bioorthogonal functional group, V represents an oxygen atom or a sulfur atom, A indicates the affinity substance, The average modification percentage r of the immunoglobulin units by the affinity substance is 65-135%. ] The affinity substance-modified antibody of [37] or a salt thereof, which contains the structural unit represented by [37].
  • the affinity substance-modified antibody or a salt thereof according to any one of [28] to [39], wherein the affinity substance-modified antibody further comprises an additional modification moiety.
  • the additional modification moiety is an additional affinity substance comprising a third affinity moiety having affinity for the constant region in the heavy chain of the antibody, and the additional affinity substance is [40] Affinity substance-modified antibody or a salt thereof, which is contained in the constant region.
  • the additional affinity substance binds to the two heavy chains through modification of the amino group in the side chain of a lysine residue present at one or more positions in the constant region of the two heavy chains.
  • the affinity substance-modified antibody of [41] or a salt thereof which is introduced into the constant region of the chain.
  • One or more positions in the constant regions of the two heavy chains are positions 246/248, 288/290, or 317 of human IgG heavy chain according to EU numbering, [42] affinity Substance-modified antibodies or salts thereof.
  • an immunoglobulin unit comprising two heavy chains and optionally two light chains, and (b) comprising a bioorthogonal functional group, and (c) the bioorthogonal functional group is An antibody derivative or a salt thereof containing a bioorthogonal functional group introduced only into the constant region of one heavy chain in an immunoglobulin unit.
  • the bioorthogonal functional group forms a constant region in the one heavy chain through modification of an amino group in the side chain of a lysine residue present at one or more positions in the constant region in the one heavy chain.
  • One or more positions in the constant region of the one heavy chain are positions 246/248, 288/290, or 317 of a human IgG heavy chain according to EU numbering, [49] or [50] antibody derivative or its salt.
  • the antibody derivative or its salt containing a bioorthogonal functional group has the following formula (IIIa): [During the ceremony, Ig indicates the immunoglobulin unit, L 1 indicates the first linker, B represents a group containing a bioorthogonal functional group, The average percentage modification r of said immunoglobulin units by bioorthogonal functional groups is between 65 and 135%.
  • the antibody derivative or its salt containing a bioorthogonal functional group has the following formula (IIIa-1): [During the ceremony, Ig indicates the immunoglobulin unit, W 1 represents an oxygen atom or a sulfur atom, L 3 indicates a third linker, SH represents a thiol group, The average percentage modification r of said immunoglobulin units by bioorthogonal functional groups is between 65 and 135%. ] The antibody derivative according to any one of [49] to [51] or a salt thereof, which contains the structural unit represented by [49] to [51].
  • the antibody derivative or its salt containing a bioorthogonal functional group has the following formula (IIIb): [During the ceremony, Ig indicates the immunoglobulin unit, L 5 indicates the fifth linker, B represents a group containing a bioorthogonal functional group, T 1 represents a monovalent group, The average percentage modification r of said immunoglobulin units by bioorthogonal functional groups is between 65 and 135%. ]
  • the antibody derivative or its salt containing a bioorthogonal functional group has the following formula (IIIb-1): [During the ceremony, Ig indicates the immunoglobulin unit, W 1 and W 2 each independently represent an oxygen atom or a sulfur atom, L 7 indicates the seventh linker, B represents a group containing a bioorthogonal functional group, T 2 represents a monovalent group, The modification percentage r of said immunoglobulin units by bioorthogonal functional groups is between 65 and 135%. ] The antibody derivative according to any one of [49] to [51] or a salt thereof, which contains the structural unit represented by [49] to [51].
  • the antibody derivative or a salt thereof according to any one of [49] to [55], wherein the antibody derivative further comprises an additional modification moiety.
  • the additional modification moiety is an additional modification moiety comprising a bioorthogonal functional group, and the additional affinity substance comprising the bioorthogonal functional group is contained in a constant region in the heavy chain of the antibody;
  • the antibody derivative of [56] or a salt thereof is present through modification of the amino group in the side chain of a lysine residue present at one or more positions in the constant region of the two heavy chains.
  • the antibody derivative of [57] or a salt thereof which is introduced into the constant regions of the two heavy chains.
  • the antibody derivative of [58] wherein one or more positions in the constant region of the two heavy chains is position 246/248, position 288/290, or position 317 of a human IgG heavy chain according to EU numbering. Or its salt.
  • an immunoglobulin unit comprising two heavy chains and optionally two light chains, and (b) a functional substance, and (c) the functional substance is in said immunoglobulin unit.
  • the functional substance is added to the constant region of the one heavy chain through modification of the amino group in the side chain of a lysine residue present at one or more positions in the constant region of the one heavy chain.
  • the conjugate of [60] or its salt, which has been introduced only in [62] One or more positions in the constant region of the one heavy chain are positions 246/248, 288/290, or 317 of a human IgG heavy chain according to EU numbering, [60] or [61] conjugates or salts thereof.
  • the conjugate or its salt has the following formula (IVa): [During the ceremony, Ig indicates the immunoglobulin unit, L 1 indicates the first linker, Z represents a functional substance; The average percentage modification r of said immunoglobulin units by functional substances is 65-135%. ] The conjugate of any one of [60] to [62] or a salt thereof, which contains a structural unit represented by [60] to [62].
  • the conjugate or its salt has the following formula (IVa-1): [During the ceremony, Ig indicates the immunoglobulin unit, W 1 represents an oxygen atom or a sulfur atom, L 3 indicates a third linker, Z represents a functional substance; The average percentage modification r of said immunoglobulin units by functional substances is 65-135%. ] The conjugate of any one of [60] to [62] or a salt thereof, which contains a structural unit represented by [60] to [62].
  • the conjugate or its salt has the following formula (IVb): [During the ceremony, Ig indicates the immunoglobulin unit, L 5 indicates the fifth linker, Z represents a functional substance; T 1 represents a monovalent group, The average percentage modification r of said immunoglobulin units by functional substances is 65-135%. ] The conjugate of any one of [60] to [62] or a salt thereof, which contains a structural unit represented by [60] to [62].
  • the conjugate or its salt has the following formula (IVb-1): [During the ceremony, Ig indicates the immunoglobulin unit, W 1 and W 2 each independently represent an oxygen atom or a sulfur atom, L 7 indicates the seventh linker, Z represents a functional substance; T 2 represents a monovalent group, The modification percentage r of said immunoglobulin units by functional substances is 65-135%. ] The conjugate of any one of [60] to [62] or a salt thereof, which contains a structural unit represented by [60] to [62]. [67] The conjugate or salt thereof according to any one of [60] to [66], wherein the functional substance is a drug, a label substance, an affinity substance, a transport substance, or a stabilizer.
  • the additional modification moiety containing the functional substance may be added to the functional substance through modification of the amino group in the side chain of a lysine residue present at one or more positions in the constant region of the two heavy chains.
  • the cleavable moiety is a cleavable moiety that can generate a bioorthogonal functional group on the antibody side by cleavage,
  • the method of [73], wherein the antibody or salt thereof that does not contain an affinity substance is an antibody derivative or a salt thereof that contains a bioorthogonal functional group.
  • the method of [74], wherein the antibody derivative or salt thereof containing a bioorthogonal functional group is any of the above antibody derivatives or salts thereof.
  • the antibody or a salt thereof that does not contain an affinity substance further comprises a bioorthogonal functional group between the antibody and the cleavable moiety, The method of [73], wherein the antibody or salt thereof that does not contain an affinity substance is an antibody derivative or a salt thereof that contains a bioorthogonal functional group. [77] The method of [76], wherein the antibody derivative or salt thereof containing a bioorthogonal functional group is any of the above antibody derivatives or salts thereof.
  • a method for producing a conjugate containing an antibody and a functional substance or a salt thereof including the following (1) and (2): (1) Producing an antibody derivative containing a bioorthogonal functional group or a salt thereof by the method of [74]; and (2) reacting an antibody derivative containing a bioorthogonal functional group or a salt thereof with a functional substance. to produce a conjugate or a salt thereof comprising an antibody and a functional substance.
  • the method of [78], wherein the conjugate or salt thereof is any of the above conjugates or salts thereof.
  • a method for producing a conjugate containing an antibody and a functional substance or a salt thereof including the following (1) and (2): (1) Producing an antibody derivative containing a bioorthogonal functional group or a salt thereof by the method of [76]; and (2) reacting an antibody derivative containing a bioorthogonal functional group or a salt thereof with a functional substance. to produce a conjugate or a salt thereof comprising an antibody and a functional substance.
  • the method of [78], wherein the conjugate or salt thereof is any of the above conjugates or salts thereof.
  • a method for producing a conjugate or a salt thereof comprising an antibody and a functional substance comprising: reacting an antibody derivative or a salt thereof comprising a bioorthogonal functional group with a functional substance to produce a conjugate or a salt thereof comprising an antibody and a functional substance;
  • the antibody derivative or salt thereof comprising a bioorthogonal functional group comprises (a) an immunoglobulin unit comprising two heavy chains and optionally two light chains; and (b) a bioorthogonal functional group; and (c) an antibody derivative or a salt thereof containing a bioorthogonal functional group, in which the bioorthogonal functional group is introduced only into the constant region of one of the heavy chains in the immunoglobulin unit;
  • a conjugate or a salt thereof comprising an antibody and a functional substance comprises (a) an immunoglobulin unit comprising two heavy chains and optionally two light chains, and (b) a functional substance, and ( c) The method is a conjugate of an antibody and a functional substance or
  • An affinity substance or a salt thereof comprising first and second affinity portions having affinity for the constant region in the heavy chain of an antibody.
  • the affinity substance has the following formula (A): AP1- LA -AP2 (A) [During the ceremony, AP1 indicates a first affinity peptide with affinity for the constant region in the heavy chain of antibodies; AP2 represents a second affinity peptide with affinity for the constant region in the heavy chain of antibodies; LA indicates a linker. ] An affinity substance for [85] or a salt thereof. [87] The affinity substance of [85] or [86] or a salt thereof, wherein the affinity substance contains only one specific reactive group.
  • the affinity substance according to any one of [85] to [87], wherein the affinity substance is an affinity polypeptide comprising first and second affinity peptides having an affinity for the constant region in the heavy chain of an antibody. sexual substances or their salts.
  • the affinity polypeptide has the following formula (A'): AP1-PL A -AP2 (A') [During the ceremony, AP1 indicates a first affinity peptide that has affinity for the constant region in the heavy chain of an antibody and is present on the N-terminal side of the affinity polypeptide; AP2 indicates a second affinity peptide that has affinity for the constant region in the heavy chain of an antibody and is present on the C-terminal side of the affinity polypeptide; PLA indicates a peptide linker.
  • any of the affinity substances [85] to [88] or a salt thereof [90]
  • the affinity polypeptide (i) contains only one amino acid residue having an amino group in its side chain, or (ii) contains an unprotected N-terminal amino group, [85] to [89] or any of its salts.
  • the affinity substance of [90] or a salt thereof, wherein the amino acid residue having an amino group in its side chain is a lysine residue.
  • the affinity substance or a salt thereof according to any one of [85] to [89], wherein the affinity polypeptide further contains a tripeptide consisting of Gln-Glu-Thr (QET) at the N-terminus.
  • One of the first and second affinity peptides is an affinity peptide that has an affinity for a constant region in the heavy chain of an antibody and has one lysine residue, and The affinity substance or the affinity substance according to any one of [85] to [93], wherein the other of the affinity peptides is an affinity peptide that has an affinity for the constant region in the heavy chain of an antibody and does not have a lysine residue. That salt.
  • a polynucleotide encoding an affinity polypeptide comprising first and second affinity peptides having affinity for the constant region in the heavy chain of an antibody.
  • a host cell comprising an expression unit comprising the polynucleotide of [95] and a promoter operably linked thereto.
  • a first modification moiety comprising a first affinity substance comprising first and second affinity parts having affinity for a constant region in an antibody heavy chain, and an affinity for a constant region in an antibody heavy chain.
  • a second modifying moiety comprising a second affinity substance comprising third and fourth affinity moieties having properties in the constant region of the heavy chain of the antibody; Affinity substance modified antibody or its salt.
  • an immunoglobulin unit comprising two heavy chains consisting of a first and a second heavy chain and optionally two light chains, and (b) the first and second modification moieties including; (c) the first modification moiety is introduced into the constant region of the first heavy chain in the immunoglobulin unit; and (d) the second modification moiety is introduced into the constant region of the first heavy chain in the immunoglobulin unit;
  • the affinity substance-modified antibody of [98] or a salt thereof is introduced into the constant region of the heavy chain of [98].
  • the first modification moiety modifies the first heavy chain by modifying an amino group in the side chain of a lysine residue present at one or more positions in the constant region of the first heavy chain.
  • one or more positions in the constant region of the first heavy chain and one or more positions in the constant region of the second heavy chain are positions 246/248 of a human IgG heavy chain according to EU numbering;
  • the affinity substance-modified antibody comprising the first and second modification moieties has the following formula (V):
  • Ig refers to an immunoglobulin unit comprising two heavy chains consisting of a first and a second heavy chain and optionally two light chains; LL and LR each independently represent a linker, A L represents the first affinity substance, AR represents the second affinity substance, The average percentage modification r L of the immunoglobulin units by the first modifying moiety and the average percentage modification r R of the immunoglobulin units by the second modifying moiety are each from 65 to 135%.
  • An affinity substance-modified antibody or a salt thereof according to any one of [98] to [101], which contains a structural unit represented by [98] to [101].
  • the antibody or salt thereof further comprises (iii') a first cleavable moiety between (i') the first affinity substance and (ii') the immunoglobulin unit, and/ or (i'') further comprising (iii'') a second cleavable moiety between the second affinity substance and (ii'') the immunoglobulin unit; Any affinity substance modified antibody or salt thereof.
  • [104] [103] Affinity substance-modified antibody or salt thereof, wherein the first and second cleavable moieties are cleavable moieties that can generate a bioorthogonal functional group on the immunoglobulin unit side by cleavage. .
  • the affinity substance-modified antibody containing the first and second modification moieties has the following formula (Va): [During the ceremony, Ig indicates the immunoglobulin unit, L L1 and L R1 each independently represent a first linker, L L2 and L R2 each independently represent a second linker, CLE(B) L and CLE(B) R each independently represent a cleavable moiety capable of generating a bioorthogonal functional group on the immunoglobulin unit side by cleavage; A L represents the first affinity substance, AR represents the second affinity substance, The average percentage modification r L of the immunoglobulin units by the first modifying moiety and the average percentage modification r R of the immunoglobulin units by the second modifying moiety are each from 65 to 135%.
  • the affinity substance-modified antibody or a salt thereof according to any one of [98] to [104], which contains a structural unit represented by [98] to [104].
  • the affinity substance-modified antibody comprising the first and second modification moieties has the following formula (Va-1): [During the ceremony, Ig indicates the immunoglobulin unit, W L1 , W L2 and W L3 , and W R1 , W R2 and W R3 each independently represent an oxygen atom or a sulfur atom, L L3 and L R3 each independently represent a third linker, L L4 and L R4 each independently represent a fourth linker, S represents a sulfur atom, A L represents the first affinity substance, AR represents the second affinity substance, The average percentage modification r L of the immunoglobulin units by the first modifying moiety and the average percentage modification r R of the immunoglobulin units by the second modifying moiety are each from 65 to 135%.
  • An affinity substance-modified antibody or a salt thereof according to any one of [98] to [104], which contains a structural unit represented by [98] to [104].
  • the antibody or salt thereof further comprises (iv') a first bioorthogonal functional group between (ii') the immunoglobulin unit and (iii') the first cleavable moiety, and/or (ii'') further comprising (iv'') a second bioorthogonal functional group between the immunoglobulin unit and (iii'') the second cleavable moiety [98]- [106] Any affinity substance-modified antibody or salt thereof.
  • the affinity substance-modified antibody containing the first and second modification moieties has the following formula (Vb): [During the ceremony, Ig indicates the immunoglobulin unit, L L5 and L R5 each independently represent a fifth linker, L L6 and L R6 each independently represent a sixth linker, BL represents a first group containing a first bioorthogonal functional group, BR represents a second group containing a second bioorthogonal functional group, CLE L and CLE R each independently represent a cleavable moiety; A L represents the first affinity substance, AR represents the second affinity substance, The average percentage modification r L of the immunoglobulin units by the first modifying moiety and the average percentage modification r R of the immunoglobulin units by the second modifying moiety are each from 65 to 135%.
  • the affinity substance-modified antibody or a salt thereof according to any one of [98] to [107], which contains a structural unit represented by [98] to [107].
  • the affinity substance-modified antibody comprising the first and second modification moieties has the following formula (Vb-1): [During the ceremony, Ig indicates the immunoglobulin unit, W L1 , W L2 and W L3 , and W R1 , W R2 and W R3 each independently represent an oxygen atom or a sulfur atom, L L7 and L R7 each independently represent a seventh linker, L L8 and L R8 each independently represent an eighth linker, BL represents a first group containing a first bioorthogonal functional group, BR represents a second group containing a second bioorthogonal functional group, V L and V R each independently represent an oxygen atom or a sulfur atom, A L represents the first affinity substance, AR represents the second affinity substance, The average percentage modification r L of the immunoglobulin units by the first modifying moiety and the
  • An affinity substance-modified antibody or a salt thereof according to any one of [98] to [108], which contains a structural unit represented by [98] to [108].
  • the antibody or salt thereof further comprises (iii') a first cleavable moiety between (i') the first affinity substance and (ii') the immunoglobulin unit, and ( ii'') further comprising (iv'') a first bioorthogonal functional group between the immunoglobulin unit and (iii'') the second cleavable moiety;
  • the affinity substance modification according to any one of [98] to [109], wherein the first cleavable moiety is a cleavable moiety that can generate a second bioorthogonal functional group on the immunoglobulin unit side by cleavage.
  • the affinity substance-modified antibody containing the first and second modification moieties has the following formula (Vc): [During the ceremony, Ig indicates the immunoglobulin unit, L R1 indicates the first linker, L R2 represents a second linker; L L5 indicates the fifth linker, L L6 indicates the sixth linker, BL represents a group containing the first bioorthogonal functional group, CLE L indicates the first cleavable portion; CLE(B) R represents a second cleavable moiety that can generate a second bioorthogonal functional group on the immunoglobulin unit side by cleavage; A L represents the first affinity substance, AR represents the second affinity substance, The average percentage modification r L of the immunoglobulin units by the first modifying moiety and the average percentage modification r R of the immunoglobulin units by the second modifying moiety are each from 65 to 135%.
  • the affinity substance-modified antibody or a salt thereof according to any one of [98] to [110], which contains a structural unit represented by [98] to [110].
  • the affinity substance-modified antibody comprising the first and second modification moieties has the following formula (Vc-1): [During the ceremony, Ig indicates the immunoglobulin unit, W L1 , W L2 and W L3 , and W R1 , W R2 and W R3 each independently represent an oxygen atom or a sulfur atom, L R3 each independently represents a third linker, L R4 each independently represents a fourth linker, L L7 each independently represents a seventh linker, L L8 each independently represents an eighth linker, BL represents a group containing the first bioorthogonal functional group, V L represents an oxygen atom or a sulfur atom, A L represents the first affinity substance, AR represents the second affinity substance, The average percentage modification r L of the immunoglobulin units by the first modifying moiety and the average percentage modification r R of the immunoglob
  • the additional modifying moiety is an additional affinity substance comprising a fifth affinity moiety having affinity for the constant region in the heavy chain of the antibody, and the additional affinity substance is [113] An affinity substance-modified antibody or a salt thereof, which is contained in the constant region.
  • the additional affinity substance binds to the two heavy chains through modification of the amino group in the side chain of a lysine residue present at one or more positions in the constant region of the two heavy chains.
  • One or more positions in the constant regions of the two heavy chains are positions 246/248, 288/290, or 317 of a human IgG heavy chain according to EU numbering, [115] affinity Substance-modified antibodies or salts thereof.
  • a method for producing an affinity substance-modified antibody or a salt thereof comprising first and second modification moieties including the following: (1) (A) a first affinity substance comprising a first and second affinity moiety having affinity for the constant region in the heavy chain of an antibody, and (B) a first reactive group for the antibody; A compound or a salt thereof is reacted with an antibody comprising an immunoglobulin unit comprising two heavy chains and optionally two light chains so that said first producing an affinity substance-modified antibody or a salt thereof comprising a first modified moiety comprising an affinity substance; and (2) comprising the first affinity substance in the constant region of the heavy chain of the immunoglobulin unit.
  • an affinity substance-modified antibody or a salt thereof a second affinity substance comprising third and fourth affinity parts having an affinity for the constant region in the heavy chain of the antibody, and (B) a second reaction against the antibody. reacting with a compound containing a functional group or a salt thereof to produce an affinity substance-modified antibody or a salt thereof comprising the first and second modification moieties in the constant region of the heavy chain in the immunoglobulin unit.
  • the affinity substance-modified antibody or salt thereof comprising the first and second modification moieties is any of the above-mentioned affinity substance-modified antibodies or salts thereof.
  • an immunoglobulin unit comprising two heavy chains consisting of a first and a second heavy chain and optionally two light chains; and (b) a first bioorthogonal functional group. a first modifying moiety comprising a second modifying moiety, and a second modifying moiety comprising a second bioorthogonal functional group; (c) a first modification moiety is introduced into the constant region of the first heavy chain; (d) a second modification moiety is introduced into the constant region of the second heavy chain, and (e) the first and second modification moieties are different from each other; Antibody derivatives or salts thereof.
  • the first modification moiety modifies the first heavy chain by modifying an amino group in the side chain of a lysine residue present at one or more positions in the constant region of the first heavy chain. Modification of an amino group in a side chain of a lysine residue that has been introduced into the constant region of the chain, and the second modification moiety is present at one or more positions in the constant region of the second heavy chain.
  • one or more positions in the constant region of the first heavy chain and one or more positions in the constant region of the second heavy chain are positions 246/248 of a human IgG heavy chain according to EU numbering;
  • the antibody derivative containing the first and second modification moieties or a salt thereof has the following formula (VIa): [During the ceremony, Ig indicates the immunoglobulin unit, L L1 and L R1 each independently represent a first linker, BL represents a first group containing a first bioorthogonal functional group, BR represents a second group containing a second bioorthogonal functional group, The average percentage modification r L of said immunoglobulin units by the first modifying moiety and the average percentage modification r R of said immunoglobulin units by the second modifying moiety are each between 65 and 135%. ] The antibody derivative of any one of [119] to [121] or a salt thereof, which contains a structural unit represented by [119] to [121].
  • the antibody derivative containing the first and second modification moieties or a salt thereof has the following formula (VIa-1): [During the ceremony, Ig indicates the immunoglobulin unit, W L1 and W R1 each independently represent an oxygen atom or a sulfur atom, L L3 and L R3 each independently represent a third linker, SH represents a thiol group, which is a bioorthogonal functional group, The average percentage modification r L of said immunoglobulin units by the first modifying moiety and the average percentage modification r R of said immunoglobulin units by the second modifying moiety are each between 65 and 135%.
  • the antibody derivative containing the first and second modification moieties or a salt thereof has the following formula (VIb): [During the ceremony, Ig indicates the immunoglobulin unit, L L5 and L R5 each independently represent a fifth linker, BL represents a first group containing a first bioorthogonal functional group, BR represents a second group containing a second bioorthogonal functional group, T L1 and T R1 each independently represent a monovalent group, The average percentage modification r L of said immunoglobulin units by the first modifying moiety and the average percentage modification r R of said immunoglobulin units by the second modifying moiety are each between 65 and 135%.
  • the antibody derivative containing the first and second modification moieties or a salt thereof has the following formula (VIb-1): [During the ceremony, Ig indicates the immunoglobulin unit, W L1 and W L2 and W R1 and W R2 each independently represent an oxygen atom or a sulfur atom, L L7 and L R7 each independently represent a seventh linker, BL represents a first group containing a first bioorthogonal functional group, BR represents a second group containing a second bioorthogonal functional group, T L2 and T R2 each independently represent a monovalent group,
  • the average percentage modification r L of said immunoglobulin units by the first modifying moiety and the average percentage modification r R of said immunoglobulin units by the second modifying moiety are each between 65 and 135%.
  • the antibody derivative containing the first and second modification moieties or a salt thereof has the following formula (VIc): [During the ceremony, Ig indicates the immunoglobulin unit, L R1 indicates the first linker, L L5 indicates the fifth linker, BL represents a first group containing a first bioorthogonal functional group, BR represents a second group containing a second bioorthogonal functional group, T L1 represents a monovalent group, The average percentage modification r L of said immunoglobulin units by the first modifying moiety and the average percentage modification r R of said immunoglobulin units by the second modifying moiety are each between 65 and 135%.
  • the antibody derivative containing the first and second modification moieties or a salt thereof has the following formula (VIc-1): [During the ceremony, Ig indicates the immunoglobulin unit, W L1 and W L2 and W R1 each independently represent an oxygen atom or a sulfur atom, L R3 represents a third linker, L L7 indicates the seventh linker, BL represents a first group containing a first bioorthogonal functional group, SH represents a thiol group, which is a second bioorthogonal functional group, T L2 represents a monovalent group,
  • the average percentage modification r L of said immunoglobulin units by the first modifying moiety and the average percentage modification r R of said immunoglobulin units by the second modifying moiety are each between 65 and 135%.
  • the additional modifying moiety is an additional modifying moiety comprising a bioorthogonal functional group, and the additional modifying moiety comprising the bioorthogonal functional group is included in a constant region in the heavy chain of the antibody; 128] or a salt thereof.
  • the additional modification moiety containing the bioorthogonal functional group is present through modification of the amino group in the side chain of a lysine residue present at one or more positions in the constant region of the two heavy chains.
  • the antibody derivative of [129] or a salt thereof which is introduced into the constant regions of the two heavy chains.
  • the antibody derivative of [130] wherein one or more positions in the constant region of the two heavy chains is position 246/248, position 288/290, or position 317 of a human IgG heavy chain according to EU numbering. Or its salt.
  • [132] (a) an immunoglobulin unit comprising two heavy chains consisting of a first and a second heavy chain and optionally two light chains; and (b) an immunoglobulin unit comprising a first functional substance. 1 and a second modifying moiety comprising a second functional substance; (c) a first modification moiety is introduced into the constant region of the first heavy chain; (d) a second modification moiety is introduced into the constant region of the second heavy chain, and (e) an antibody and the first and second modifications, wherein the first and second modification moieties are different from each other; conjugates of parts or their salts.
  • the first modification moiety modifies the first heavy chain by modifying an amino group in the side chain of a lysine residue present at one or more positions in the constant region of the first heavy chain. Modification of an amino group in a side chain of a lysine residue that has been introduced into the constant region of the chain, and the second modification moiety is present at one or more positions in the constant region of the second heavy chain.
  • one or more positions in the constant region of the first heavy chain and one or more positions in the constant region of the second heavy chain are positions 246/248 of a human IgG heavy chain according to EU numbering; A conjugate of [132] or [133], or a salt thereof, at a position selected from the group consisting of positions 288/290 and 317, and combinations thereof.
  • the conjugate or its salt has the following formula (VIIa): [During the ceremony, Ig indicates the immunoglobulin unit, L L1 and L R1 each independently represent a first linker, ZL represents the first functional substance, Z R represents a second functional substance, The average percentage modification r L of said immunoglobulin units by the first modifying moiety and the average percentage modification r R of said immunoglobulin units by the second modifying moiety are each between 65 and 135%. ] The conjugate of any one of [132] to [134] or a salt thereof, which contains a structural unit represented by [132] to [134].
  • the conjugate or its salt has the following formula (VIIa-1): [During the ceremony, Ig indicates the immunoglobulin unit, W L1 and W R1 each independently represent an oxygen atom or a sulfur atom, L L3 and L R3 each independently represent a third linker, ZL represents the first functional substance, Z R represents a second functional substance, The average percentage modification r L of said immunoglobulin units by the first modifying moiety and the average percentage modification r R of said immunoglobulin units by the second modifying moiety are each between 65 and 135%. ] A conjugate of [132] to [135] or a salt thereof, which contains a structural unit represented by [132] to [135].
  • the conjugate or its salt has the following formula (VIIb): [During the ceremony, Ig indicates the immunoglobulin unit, L L5 and L R5 each independently represent a fifth linker, ZL represents the first functional substance, Z R represents a second functional substance, T L1 and T R1 each independently represent a monovalent group, The average percentage modification r L of said immunoglobulin units by the first modifying moiety and the average percentage modification r R of said immunoglobulin units by the second modifying moiety are each between 65 and 135%. ] A conjugate of [132] to [136] or a salt thereof, which contains a structural unit represented by [132] to [136].
  • the conjugate or its salt has the following formula (VIIb-1): [During the ceremony, Ig indicates the immunoglobulin unit, W L1 and W L2 and W R1 and W R2 each independently represent an oxygen atom or a sulfur atom, L L7 and L R7 each independently represent a seventh linker, ZL represents the first functional substance, Z R represents a second functional substance, T L2 and T R2 each independently represent a monovalent group, The average percentage modification r L of said immunoglobulin units by the first modifying moiety and the average percentage modification r R of said immunoglobulin units by the second modifying moiety are each between 65 and 135%.
  • the conjugate or its salt has the following formula (VIIc): [During the ceremony, Ig indicates the immunoglobulin unit, L R1 indicates the first linker, L L5 indicates the fifth linker, ZL represents the first functional substance, Z R represents a second functional substance, T L1 represents a monovalent group, The average percentage modification r L of said immunoglobulin units by the first modifying moiety and the average percentage modification r R of said immunoglobulin units by the second modifying moiety are each between 65 and 135%.
  • the conjugate or its salt has the following formula (VIIc-1): [During the ceremony, Ig indicates the immunoglobulin unit, W L1 and W L2 and W R1 each independently represent an oxygen atom or a sulfur atom, L R3 represents a third linker, L L7 indicates the seventh linker, ZL represents the first functional substance, Z R represents a second functional substance, T L2 represents a monovalent group, The average percentage modification r L of said immunoglobulin units by the first modifying moiety and the average percentage modification r R of said immunoglobulin units by the second modifying moiety are each between 65 and 135%.
  • One of the first and second functional substances is a full-length antibody or a fragment thereof, or both the first and second functional substances are each independently a full-length antibody or a fragment thereof.
  • the conjugate of [132] to [142] or a salt thereof, wherein the conjugate further comprises an additional modification moiety.
  • the additional modification moiety containing the functional substance may be added to the functional substance through modification of the amino group in the side chain of a lysine residue present at one or more positions in the constant region of the two heavy chains.
  • the conjugate of [145] wherein one or more positions in the constant regions of the two heavy chains is position 246/248, position 288/290, or position 317 of a human IgG heavy chain according to EU numbering. Or its salt.
  • only one heavy chain in an antibody structural unit (an immunoglobulin unit containing two heavy chains) can be easily modified. Further, according to the present invention, it is possible to easily modify only one heavy chain in the structural unit of the antibody and provide a position-selectively modified antibody.
  • FIG. 1 is a schematic diagram showing modification of the structural unit of an antibody by the compound of the present invention represented by formula (I) or a salt thereof.
  • FIG. 2 is a diagram showing an overview of one embodiment of the present invention.
  • FIG. 3 is a diagram illustrating an overview of another embodiment of the invention.
  • FIG. 4 is a diagram showing an overview of yet another embodiment of the present invention.
  • FIG. 5 is a diagram showing an overview of yet another embodiment of the present invention.
  • FIG. 6 is a diagram showing an overview of yet another embodiment of the present invention.
  • FIG. 7 is a diagram showing an overview of an embodiment of the present invention.
  • FIG. 8 is a diagram showing an overview of another embodiment of the present invention.
  • FIG. 9 is a diagram showing an overview of yet another embodiment of the present invention.
  • FIG. 1 is a schematic diagram showing modification of the structural unit of an antibody by the compound of the present invention represented by formula (I) or a salt thereof.
  • FIG. 2 is a diagram showing an overview of one embodiment of
  • FIG. 10 is a diagram showing an overview of yet another embodiment of the present invention.
  • FIG. 11 is a diagram showing an overview of yet another embodiment of the present invention.
  • FIG. 12 is a diagram showing an overview of yet another embodiment of the present invention.
  • FIG. 13 is a diagram showing an overview of yet another embodiment of the present invention.
  • FIG. 14 is a diagram showing an overview of yet another embodiment of the present invention.
  • FIG. 15 is a diagram showing an overview of yet another embodiment of the present invention.
  • FIG. 16 is a diagram showing the expression of each polypeptide in each transformant.
  • FIG. 17-1 is a diagram showing a sensorgram showing the affinity of the polypeptide QET-Z34CM-PA32-Fc3K (SEQ ID NO: 1) and the IgG1 antibody Fc region.
  • FIG. 17-1 is a diagram showing a sensorgram showing the affinity of the polypeptide QET-Z34CM-PA32-Fc3K (SEQ ID NO: 1) and the IgG1 antibody Fc
  • FIG. 17-2 is a diagram showing a sensorgram showing the affinity of the polypeptide QET-Z34CM-PA48-Fc3K (SEQ ID NO: 2) and the IgG1 antibody Fc region.
  • FIG. 17-3 is a diagram showing a sensorgram showing the affinity of the polypeptide QET-Fc3-PA32-Z34CM (SEQ ID NO: 3) and the IgG1 antibody Fc region.
  • FIG. 17-4 is a diagram showing a sensorgram showing the affinity of the polypeptide QET-Fc3K-PA48-Z34CM (SEQ ID NO: 4) and the IgG1 antibody Fc region.
  • FIG. 17-5 is a diagram showing a sensorgram showing the affinity of the polypeptide QET-Fc3K-PA32-ProAR (SEQ ID NO: 5) and the IgG1 antibody Fc region.
  • FIG. 17-6 is a diagram showing a sensorgram showing the affinity of the polypeptide QET-Fc3K-PA48-ProAR (SEQ ID NO: 6) and the IgG1 antibody Fc region.
  • FIG. 17-7 is a diagram showing a sensorgram showing the affinity of the polypeptide QET-ProAR-PA32-Z34CK (SEQ ID NO: 7) and the IgG1 antibody Fc region.
  • FIG. 17-8 is a diagram showing a sensorgram showing the affinity of the polypeptide QET-ProAR-PA48-Z34CK (SEQ ID NO: 8) and the IgG1 antibody Fc region.
  • FIG. 18 is a diagram showing the results of confirmation of the peptide/antibody binding ratio.
  • FIG. 19-1 is a diagram showing modification sites of lysine residues in the constant region of the heavy chain of an antibody by LC-MS/MS.
  • FIG. 19-2 is a diagram showing modification of the lysine residue at position 246 or 248 of the heavy chain according to EU numbering by CID spectrum.
  • FIG. 19-3 is a diagram showing the selectivity of modification to the lysine residue at position 248 by BioPharma Finder.
  • FIG. 19-1 is a diagram showing modification sites of lysine residues in the constant region of the heavy chain of an antibody by LC-MS/MS.
  • FIG. 19-2 is a diagram showing modification of the lysine residue at position 246 or
  • FIG. 20-1 is a sensorgram showing the affinity between EGFR and cetuximab.
  • Figure 20-2 is a sensorgram showing the affinity of fetal Fc receptor (FcRn) and cetuximab.
  • FIG. 20-3 is a sensorgram showing the pH-dependent affinity between FcRn and cetuximab.
  • FIG. 21-1 is a sensorgram showing the affinity between EGFR and bi-specific antibody (Cetuximab-Trastuzumab Fab) (M-3).
  • FIG. 21-2 is a sensorgram showing the affinity between HER2 and bi-specific antibody (Cetuximab-Trastuzumab Fab) (M-3).
  • FIG. 21-3 is a sensorgram showing the affinity between FcRn and bi-specific antibody (Cetuximab-Trastuzumab Fab) (M-3).
  • FIG. 21-4 is a sensorgram showing the pH-dependent affinity between FcRn and bi-specific antibody (Cetuximab-Trastuzumab Fab) (M-3).
  • FIG. 22-1 is a sensorgram showing the affinity between EGFR and bi-specific antibody (Trastuzumab-Cetuximab Fab) (M-4).
  • FIG. 22-2 is a sensorgram showing the affinity between HER2 and bi-specific antibody (Trastuzumab-Cetuximab Fab) (M-4).
  • FIG. 22-3 is a sensorgram showing the affinity between FcRn and bi-specific antibody (Trastuzumab-Cetuximab Fab) (M-4).
  • FIG. 22-4 is a sensorgram showing the pH-dependent affinity between FcRn and bi-specific antibody (Trastuzumab-Cetuximab Fab) (M-4).
  • FIG. 23-1 is a sensorgram showing the affinity between EGFR and the tri-specific antibody (M-2).
  • FIG. 23-2 is a sensorgram showing the affinity between HER2 and the tri-specific antibody (M-2).
  • FIG. 23-3 is a sensorgram showing the affinity between PD-1 and the tri-specific antibody (M-2).
  • FIG. 23-4 is a sensorgram showing the affinity between FcRn and Tri-specific antibody (M-2).
  • FIG. 23-5 is a sensorgram showing the pH-dependent affinity between FcRn and Tri-specific antibody (M-2).
  • Figure 24-1 shows cetuximab (Cmab), trastuzumab (Tmab), bi-specific antibody (Cetuximab-Trastuzumab Fab) (M-3) (Trastuzumab-Cetuximab Fab) (M-4) calculated by flow cytometry.
  • FIG. 2 is a diagram showing the positive rate of SKBR-3 cells (HER2 positive) and A-431 cells (EGFR positive) to which tri-specific antibody (Pembrolizumab Fab-Cetuximab-Trastuzumab Fab) (M-2) was bound.
  • Figure 24-2 is a diagram showing evaluation of cetuximab (Cmab) binding to SKBR-3 cells (HER2 positive), A-431 cells (EGFR positive) and T cells (PD-1 positive) by flow cytometry.
  • FIG. 24-3 is a diagram showing evaluation of the binding of trastuzumab (Tmab) to SKBR-3 cells (HER2 positive) and A-431 cells (EGFR positive) by flow cytometry.
  • Figure 24-4 shows evaluation of binding of bi-specific antibody (Cetuximab-Trastuzumab Fab) (M-3) to SKBR-3 cells (HER2 positive) and A-431 cells (EGFR positive) by flow cytometry. It is.
  • Figure 24-5 shows evaluation of binding of bi-specific antibody (Trastuzumab-Cetuximab Fab) (M-4) to SKBR-3 cells (HER2 positive) and A-431 cells (EGFR positive) by flow cytometry.
  • FIG. 24-6 is a diagram showing evaluation of binding of pembrolizumab (Pbl) to T cells (PD-1 positive) by flow cytometry.
  • FIG. 24-7 shows tri-specific antibodies (Pembrolizumab Fab-Cetuximab-Trastuzumab Fab) against SKBR-3 cells (HER2 positive), A-431 cells (EGFR positive) and T cells (PD-1 positive) by flow cytometry.
  • M-2 is a diagram showing the evaluation of the bond.
  • Figure 24-8 shows the T cells (PD-1 It is a figure showing the positive rate of positive).
  • FIG. 25 shows SDS-PAGE of CD3-VHH-PA24H6-AzF (V-1) expression.
  • FIG. 26 is a diagram showing the results of evaluating the affinity of CD3-VHH (V-1).
  • FIG. 27 is a diagram showing the results of evaluating the affinity of CD3-VHH (V-1).
  • FIG. 28 is a diagram showing SDS-PAGE of six types of affinity peptides.
  • the term “antibody” is as follows.
  • immunoglobulin unit corresponds to a bivalent monomeric unit that is a constituent unit of such an antibody, and includes two heavy chains and optionally two light chains. It is an immunoglobulin unit. Therefore, terms and expressions regarding immunoglobulin units such as their origin, type (polyclonal or monoclonal, isotype, and full-length antibody or antibody fragment), antigen, position of amino acid residues (e.g., lysine residues), and regioselectivity, etc. The definitions, examples and preferred examples of are similar to those of antibodies described below and are used interchangeably with the expression "antibody”.
  • the origin of the antibody is not particularly limited, and may be derived from animals such as mammals and birds (eg, chickens).
  • the immunoglobulin unit is derived from a mammal.
  • mammals include, for example, primates (e.g., humans, monkeys, chimpanzees), rodents (e.g., mice, rats, guinea pigs, hamsters, rabbits), companion animals (e.g., dogs, cats), and domestic animals. (eg, cows, pigs, goats), working animals (eg, horses, sheep), preferably primates or rodents, and more preferably humans.
  • the type of antibody may be a polyclonal antibody or a monoclonal antibody.
  • the antibody may also be a divalent antibody (eg, IgG, IgD, IgE) or a quadrivalent or higher antibody (eg, IgA antibody, IgM antibody).
  • Preferably the antibody is a monoclonal antibody.
  • Monoclonal antibodies include, for example, chimeric antibodies, humanized antibodies, human antibodies, antibodies to which predetermined sugar chains have been added (e.g., antibodies modified to have a sugar chain binding consensus sequence such as an N-type sugar chain binding consensus sequence) antibodies), bispecific antibodies, Fc region proteins, Fc fusion proteins, and disulfide bond reduced antibodies.
  • Isotypes of monoclonal antibodies include, for example, IgG (eg, IgG1, IgG2, IgG3, IgG4), IgM, IgA, IgD, IgE, and IgY.
  • IgG eg, IgG1, IgG2, IgG3, IgG4
  • IgM IgA, IgD, IgE, and IgY.
  • full-length antibodies or antibody fragments containing variable regions and CH1 and CH2 domains can be used as monoclonal antibodies, but full-length antibodies are preferred.
  • the antibody is preferably a human IgG monoclonal antibody, more preferably a human IgG full-length monoclonal antibody.
  • any antigen can be used as the antigen for the antibody.
  • antigens include proteins [oligopeptides and polypeptides]. Examples include proteins modified with biomolecules such as sugars (eg, glycoproteins)], sugar chains, nucleic acids, and low-molecular compounds.
  • the antibody may be an antibody that uses a protein as an antigen. Examples of proteins include cell membrane receptors, cell membrane proteins other than cell membrane receptors (eg, extracellular matrix proteins), ligands, and soluble receptors.
  • the protein that is the antigen of the antibody may be a disease target protein.
  • diseases target proteins include the following.
  • CGRP Cranial nerve diseases CGRP, CD20, ⁇ -amyloid, ⁇ -amyloid protofibrin, Calcitonin Gene-Related Peptide Receptor, LINGO (Ig Domain Containing 1), ⁇ -synuclein, extracellular tau, CD52, insulin receptor, ta u protein, TDP-43 , SOD1, TauC3, JC virus
  • Amyloid AL SEMA4D (CD100), insulin receptor, ANGPTL3, IL4, IL13, FGF23, adrenocorticotropic hormone, transthyretin, huntingtin
  • monoclonal antibodies include certain chimeric antibodies (e.g., rituximab, basiliximab, infliximab, cetuximab, siltuximab, dinutuximab, ortatoximab), certain humanized antibodies (e.g., daclizumab, palivizumab, trastuzumab, alentuzumab, omalizumab).
  • chimeric antibodies e.g., rituximab, basiliximab, infliximab, cetuximab, siltuximab, dinutuximab, ortatoximab
  • humanized antibodies e.g., daclizumab, palivizumab, trastuzumab, alentuzumab, omalizumab.
  • efalizumab bevacizumab, natalizumab (IgG4), tocilizumab, eculizumab (IgG2), mogamlizumab, pertuzumab, obinutuzumab, vedrizumab, penprolizumab (IgG4), mepolizumab, elotuzumab, daratumumab, ikeseki horrab (IgG4), reslizumab (IgG4), atezolizumab), specific human antibodies (e.g., adalimumab (IgG1), panitumumab, golimumab, ustekinumab, canakinumab, ofatumumab, denosumab (IgG2), ipilimumab, belimumab, raxibacumab, ramucirumab, nivolumab, dupiluma
  • specific amino acid residues in the constant region of the heavy chain of an antibody can be selectively modified.
  • specific amino acid residues include, for example, lysine residues, tyrosine residues, serine residues, and threonine residues.
  • human IgG such as human IgG1
  • the following amino acid residues present in the heavy chain constant region can be exposed on the antibody surface, so these amino acid residues can be used to introduce a specific cleavage site ( The positions of amino acid residues are according to EU numbering; see http://www.imgt.org/IMGTScientificChart/Numbering/Hu_IGHGnber.html).
  • Exposed lysine residues CH2 domain e.g., positions 246, 248, 274, 288, 290, 317, 320, 322) CH3 domain (e.g., positions 360, 414, 439)
  • Exposed tyrosine residues in CH2 domain e.g., positions 278, 296, and 300
  • CH3 domain e.g., position 436
  • Exposed serine residue CH2 domain e.g., positions 254, 267, 298
  • CH3 domain e.g., position 400, position 415, position 440
  • Exposed threonine residue CH2 domain e.g., positions 256 and 289) CH3 domain (e.g., positions 335 and 359)
  • the positions of amino acid residues in antibodies and the positions of heavy chain constant regions follow EU numbering (see http://www.imgt.org/IMGTScientificChart/Numbering/Hu_IGHGnber.html).
  • the lysine residue at position 246 corresponds to the 16th amino acid residue of the human IgG CH2 region
  • the lysine residue at position 248 corresponds to the 18th amino acid residue of the human IgG CH2 region.
  • the lysine residue at position 288 corresponds to the 58th amino acid residue of the human IgG CH2 region
  • the lysine residue at position 290 corresponds to the 60th amino acid residue of the human IgG CH2 region.
  • the lysine residue at position 317 corresponds to the 87th amino acid residue of the human IgG CH2 region.
  • the notation 246/248 indicates that the lysine residue at position 246 or 248 is of interest.
  • the notation 288/290 indicates that the lysine residue at position 288 or 290 is of interest.
  • the specific amino acid residue in the constant region of the heavy chain that is regioselectively modified is a lysine residue (e.g., a lysine residue at positions 246/248 or 288/290). can do.
  • regioselective or “regioselectivity” refers to binding to a specific amino acid residue in an antibody even though the specific amino acid residue is not unevenly distributed in a specific region of the antibody. This means that certain structural units that can be formed are unevenly distributed in a specific region of an antibody. Therefore, expressions related to regioselectivity such as “regioselectively possessing", “regioselective binding”, “regioselective binding”, etc.
  • the retention rate or binding rate of a predetermined structural unit in the target region is more significant than the retention rate or binding rate of the structural unit in a non-target region containing multiple amino acid residues that are the same as the specific amino acid residue in the target region. It means high level.
  • Such regioselectivity is 50% or more, preferably 60% or more, more preferably 70% or more, even more preferably 80% or more, particularly preferably 90% or more, 95% or more, 96% or more, It may be 97% or more, 98% or more, 99% or more, 99.5% or more, or 100%.
  • specific lysine residues in the heavy chain of an antibody can be selectively modified without using a peptide-containing linker.
  • Peptide moieties have potential immunogenicity and are also susceptible to hydrolysis in blood. Therefore, avoidance of the use of linkers containing peptide moieties is desirable in clinical applications.
  • a specific amino acid residue in the constant region of a heavy chain e.g., a lysine residue at a specific position
  • specific amino acid residues at other positions are further regioselectively modified.
  • methods for position-selectively modifying specific amino acid residues at predetermined positions in antibodies are disclosed in WO 2018/199337, WO 2019/240288, WO 2019/240287, and WO It is described in No. 2020/090979.
  • Such specific amino acid residues include amino acid residues (e.g., lysine residues, aspartic acid residues) that have side chains that are easily modified (e.g., amino groups, carboxy groups, amide groups, hydroxy groups, thiol groups). , glutamic acid residues, asparagine residues, glutamine residues, threonine residues, serine residues, tyrosine residues, cysteine residues), but preferably lysine residues having side chains containing amino groups, hydroxy Tyrosine, serine, and threonine residues with side chains containing groups, or cysteine residues with side chains containing thiol groups, and more preferably may be lysine residues (i.e.
  • lysine residues at positions 246/248 lysine residues at 288/290, and lysine residues at position 317
  • two lysine residues may be double-modified in a position-selective manner, and three Lysine residues may be regioselectively triple modified).
  • halogen atom examples include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom.
  • Examples of the monovalent group include a monovalent hydrocarbon group and a monovalent heterocyclic group.
  • the monovalent group is one or more (for example, 1 to 10, preferably 1 to 8, more preferably 1 to 6, even more preferably 1 to 5, particularly preferably 1 to 3) as described below. may be substituted with a substituent.
  • Examples of the monovalent hydrocarbon group include a monovalent chain hydrocarbon group, a monovalent alicyclic hydrocarbon group, and a monovalent aromatic hydrocarbon group.
  • a monovalent chain hydrocarbon group means a hydrocarbon group composed only of a chain structure, and does not include a cyclic structure in the main chain. However, the chain structure may be linear or branched. Examples of the monovalent chain hydrocarbon group include alkyl, alkenyl, and alkynyl. Alkyl, alkenyl, and alkynyl may be linear or branched.
  • the alkyl is preferably an alkyl having 1 to 12 carbon atoms, more preferably an alkyl having 1 to 6 carbon atoms, and even more preferably an alkyl having 1 to 4 carbon atoms.
  • alkyl having 1 to 12 carbon atoms include methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, isobutyl, t-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl. , dodecyl.
  • the alkenyl is preferably an alkenyl having 2 to 12 carbon atoms, more preferably an alkenyl having 2 to 6 carbon atoms, and even more preferably an alkenyl having 2 to 4 carbon atoms.
  • alkenyl has a substituent, the number of carbon atoms of the substituent is not included in the above number of carbon atoms.
  • alkenyl having 2 to 12 carbon atoms include vinyl, propenyl, and n-butenyl.
  • alkynyl alkynyl having 2 to 12 carbon atoms is preferred, alkynyl having 2 to 6 carbon atoms is more preferred, and alkynyl having 2 to 4 carbon atoms is even more preferred.
  • alkynyl has a substituent, the number of carbon atoms of the substituent is not included in the above number of carbon atoms.
  • alkynyl having 2 to 12 carbon atoms include ethynyl, propynyl, and n-butynyl.
  • alkyl is preferable.
  • a monovalent alicyclic hydrocarbon group means a hydrocarbon group that contains only an alicyclic hydrocarbon as a ring structure and does not contain an aromatic ring. It may be. However, it is not necessary to be composed only of alicyclic hydrocarbons, and a part thereof may contain a chain structure. Examples of the monovalent alicyclic hydrocarbon group include cycloalkyl, cycloalkenyl, and cycloalkynyl, and these may be monocyclic or polycyclic.
  • cycloalkyl As cycloalkyl, cycloalkyl having 3 to 12 carbon atoms is preferred, cycloalkyl having 3 to 6 carbon atoms is more preferred, and cycloalkyl having 5 to 6 carbon atoms is even more preferred.
  • cycloalkyl has a substituent, the number of carbon atoms of the substituent is not included in the above number of carbon atoms.
  • Examples of cycloalkyl having 3 to 12 carbon atoms include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.
  • cycloalkenyl having 3 to 12 carbon atoms is preferred, cycloalkenyl having 3 to 6 carbon atoms is more preferred, and cycloalkenyl having 5 to 6 carbon atoms is even more preferred.
  • cycloalkenyl has a substituent, the number of carbon atoms of the substituent is not included in the number of carbon atoms.
  • Examples of cycloalkenyl having 3 to 12 carbon atoms include cyclopropenyl, cyclobutenyl, cyclopentenyl, and cyclohexenyl.
  • cycloalkynyl As cycloalkynyl, cycloalkynyl having 3 to 12 carbon atoms is preferred, cycloalkynyl having 3 to 6 carbon atoms is more preferred, and cycloalkynyl having 5 to 6 carbon atoms is even more preferred.
  • cycloalkynyl has a substituent, the number of carbon atoms of the substituent is not included in the above number of carbon atoms.
  • Examples of cycloalkynyl having 3 to 12 carbon atoms include cyclopropynyl, cyclobutynyl, cyclopentynyl, and cyclohexynyl.
  • cycloalkyl is preferable.
  • a monovalent aromatic hydrocarbon group means a hydrocarbon group containing an aromatic ring structure. However, it does not have to be composed only of aromatic rings, and may include a chain structure or alicyclic hydrocarbon as part of it, and the aromatic ring may be monocyclic or polycyclic. good.
  • the monovalent aromatic hydrocarbon group is preferably an aryl having 6 to 12 carbon atoms, more preferably an aryl having 6 to 10 carbon atoms, and even more preferably an aryl having 6 carbon atoms.
  • the monovalent aromatic hydrocarbon group has a substituent, the number of carbon atoms of the substituent is not included in the above number of carbon atoms. Examples of aryl having 6 to 12 carbon atoms include phenyl and naphthyl.
  • phenyl is preferred.
  • alkyl, cycloalkyl, and aryl are preferable as the monovalent hydrocarbon group.
  • a monovalent heterocyclic group refers to a group obtained by removing one hydrogen atom from the heterocycle of a heterocyclic compound.
  • the monovalent heterocyclic group is a monovalent aromatic heterocyclic group or a monovalent non-aromatic heterocyclic group.
  • the heteroatom constituting the heterocyclic group preferably contains one or more selected from the group consisting of an oxygen atom, a sulfur atom, a nitrogen atom, a phosphorus atom, a boron atom, and a silicon atom; It is more preferable that at least one type selected from the group consisting of atoms is included.
  • the monovalent aromatic heterocyclic group is preferably an aromatic heterocyclic group having 1 to 15 carbon atoms, more preferably an aromatic heterocyclic group having 1 to 9 carbon atoms, and an aromatic heterocyclic group having 1 to 6 carbon atoms.
  • Group heterocyclic groups are more preferred.
  • the monovalent aromatic heterocyclic group has a substituent, the number of carbon atoms of the substituent is not included in the above number of carbon atoms.
  • Examples of monovalent aromatic heterocyclic groups include pyrrolyl, furanyl, thiophenyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, pyrazolyl, imidazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, triazolyl, tetrazolyl, indolyl, purinyl, and anthraquinolyl. , carbazonyl, fluorenyl, quinolinyl, isoquinolinyl, quinazolinyl, and phthalazinyl.
  • the monovalent non-aromatic heterocyclic group is preferably a non-aromatic heterocyclic group having 2 to 15 carbon atoms, more preferably a non-aromatic heterocyclic group having 2 to 9 carbon atoms, and preferably a non-aromatic heterocyclic group having 2 to 9 carbon atoms.
  • the non-aromatic heterocyclic group of 6 is more preferred.
  • the monovalent non-aromatic heterocyclic group has a substituent, the number of carbon atoms of the substituent is not included in the above number of carbon atoms.
  • Examples of the monovalent non-aromatic heterocyclic group include oxiranyl, aziridinyl, azetidinyl, oxetanyl, thietanyl, pyrrolidinyl, dihydrofuranyl, tetrahydrofuranyl, dioxolanyl, tetrahydrothiophenyl, pyrrolinyl, imidazolidinyl, oxazolidinyl, piperidinyl, dihydropyranyl.
  • tetrahydropyranyl tetrahydrothiopyranyl
  • morpholinyl thiomorpholinyl
  • piperazinyl dihydroxazinyl, tetrahydroxazinyl, dihydropyrimidinyl, and tetrahydropyrimidinyl.
  • the monovalent heterocyclic group is preferably a 5- or 6-membered heterocyclic group.
  • R 1 represents a hydrogen atom or a substituent described below.
  • R 2 represents a divalent linear hydrocarbon group, a divalent cyclic hydrocarbon group, or a divalent heterocyclic group.
  • n and m are each an integer of 1 to 10, preferably an integer of 1 to 8, more preferably an integer of 1 to 6, even more preferably an integer of 1 to 5, particularly preferably is an integer from 1 to 3.
  • the divalent straight-chain hydrocarbon group is straight-chain alkylene, straight-chain alkenylene, or straight-chain alkynylene.
  • the straight chain alkylene is a straight chain alkylene having 1 to 6 carbon atoms, preferably a straight chain alkylene having 1 to 4 carbon atoms. Examples of linear alkylene include methylene, ethylene, n-propylene, n-butylene, n-pentylene, and n-hexylene.
  • Straight chain alkenylene is straight chain alkenylene having 2 to 6 carbon atoms, preferably straight chain alkenylene having 2 to 4 carbon atoms.
  • linear alkenylene examples include ethylenylene, n-propynylene, n-butenylene, n-pentenylene, and n-hexenylene.
  • Straight chain alkynylene is straight chain alkynylene having 2 to 6 carbon atoms, preferably straight chain alkynylene having 2 to 4 carbon atoms.
  • linear alkynylene examples include ethynylene, n-propynylene, n-butynylene, n-pentynylene, and n-hexynylene.
  • straight chain alkylene is preferred.
  • the divalent cyclic hydrocarbon group is arylene or a divalent non-aromatic cyclic hydrocarbon group.
  • arylene arylene having 6 to 14 carbon atoms is preferred, arylene having 6 to 10 carbon atoms is more preferred, and arylene having 6 carbon atoms is particularly preferred.
  • Examples of arylene include phenylene, naphthylene, and anthracenylene.
  • the divalent non-aromatic cyclic hydrocarbon group is preferably a monocyclic or polycyclic divalent non-aromatic cyclic hydrocarbon group having 3 to 12 carbon atoms; A cyclic or polycyclic divalent non-aromatic cyclic hydrocarbon group is more preferred, and a monocyclic divalent non-aromatic cyclic hydrocarbon group having 5 to 8 carbon atoms is particularly preferred.
  • Examples of the divalent non-aromatic cyclic hydrocarbon group include cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, and cyclooctylene.
  • arylene is preferable.
  • the divalent heterocyclic group is a divalent aromatic heterocyclic group or a divalent non-aromatic heterocyclic group.
  • the heteroatom constituting the heterocycle preferably contains one or more selected from the group consisting of an oxygen atom, a sulfur atom, a nitrogen atom, a phosphorus atom, a boron atom, and a silicon atom; It is more preferable to include one or more selected from the group consisting of:
  • the divalent aromatic heterocyclic group is preferably a divalent aromatic heterocyclic group having 3 to 15 carbon atoms, more preferably a divalent aromatic heterocyclic group having 3 to 9 carbon atoms. Particularly preferred are divalent aromatic heterocyclic groups having numbers 3 to 6.
  • divalent aromatic heterocyclic group examples include pyrrolediyl, furandiyl, thiophenediyl, pyridinediyl, pyridazinediyl, pyrimidinediyl, pyrazinediyl, triazinediyl, pyrazolediyl, imidazolediyl, thiazolediyl, isothiazolediyl, oxazoldiyl.
  • the divalent non-aromatic heterocyclic group is preferably a non-aromatic heterocyclic group having 3 to 15 carbon atoms, more preferably a non-aromatic heterocyclic group having 3 to 9 carbon atoms, and is more preferably a non-aromatic heterocyclic group having 3 to 9 carbon atoms.
  • the non-aromatic heterocyclic group of 6 is particularly preferred.
  • Examples of the divalent non-aromatic heterocyclic group include pyrroledionediyl, pyrrolinedionediyl, oxiranediyl, aziridinediyl, azetidinediyl, oxetanediyl, thietanediyl, pyrrolidinediyl, dihydrofurandiyl, tetrahydrofurandiyl, dioxolandiyl, and tetrahydrothiophene.
  • divalent heterocyclic group a divalent aromatic heterocyclic group is preferred.
  • the main chain structure of the divalent group has one or more (for example, 1 to 10, preferably 1 to 8, more preferably 1 to 6, even more preferably 1 to 5, particularly preferably 1 to 3) ) may be substituted with a substituent described below.
  • Aralkyl refers to arylalkyl.
  • the definitions, examples and preferred examples of aryl and alkyl in arylalkyl are as described above.
  • the aralkyl is preferably an aralkyl having 3 to 15 carbon atoms. Examples of such aralkyl include benzoyl, phenethyl, naphthylmethyl, and naphthylethyl.
  • Bioorthogonal functional group does not react with biological components (e.g., amino acids, proteins, nucleic acids, lipids, sugars, phosphates), or has a slow reaction rate with biological components, but does not react with biological components other than biological components.
  • biological components e.g., amino acids, proteins, nucleic acids, lipids, sugars, phosphates
  • a group that reacts selectively with Bioorthogonal functional groups are well known in the art (e.g., Sharpless K. B. et al., Angew. Chem. Int. Ed. 40, 2004 (2015); Bertozzi C. R. et al., Science 291, 2357 (2001); see Bertozzi C.R. et al., Nature Chemical Biology 1, 13 (2005)).
  • a bioorthogonal functional group for proteins is used as the bioorthogonal functional group. This is because the antibodies to be derivatized with the reagents of the present invention are proteins.
  • a bioorthogonal functional group for a protein is a group that does not react with the side chains of the 20 natural amino acid residues that make up the protein, or a group that reacts with the desired functional group although the rate of reaction with the side chain is slow. .
  • the 20 natural amino acids that make up proteins are alanine (A), asparagine (N), cysteine (C), glutamine (Q), glycine (G), isoleucine (I), leucine (L), and methionine (M).
  • glycine has no side chain (i.e., is a hydrogen atom), and glycine has a side chain that is a hydrocarbon group (i.e., from the group consisting of sulfur, nitrogen, and oxygen atoms).
  • bioorthogonal functional groups for proteins include asparagine, glutamine, methionine, proline, serine, threonine, tryptophan, tyrosine, in addition to the side chains of those amino acids that have side chains that are inert to normal reactions. , aspartic acid, glutamic acid, arginine, histidine, and lysine, which do not react with the side chains or react at a slow rate, but react with the desired functional group.
  • bioorthogonal functional groups include, for example, azide residues, aldehyde residues, thiol residues, alkene residues (in other words, vinylene (ethenylene) moieties, which are the smallest units having double bonds between carbon atoms).
  • alkyne residue in other words, it is sufficient to have an ethynylene moiety, which is the smallest unit having a triple bond between carbon atoms; the same applies hereinafter
  • halogen residue tetrazine residue, nitrone residue, hydroxylamine residue, nitrile residue, hydrazine residue, ketone residue, boronic acid residue, cyanobenzothiazole residue, allyl residue, phosphine residue, maleimide residue, disulfide residue , thioester residue, ⁇ -halocarbonyl residue (e.g., carbonyl residue having a fluorine atom, chlorine atom, bromine atom or iodine atom at the ⁇ position; the same applies hereinafter), isonitrile residue, sydone residue, selenium residue. Can be mentioned.
  • the bioorthogonal functional group may be protected or unprotected.
  • a bioorthogonal functional group refers to an unprotected bioorthogonal functional group or a protected bioorthogonal functional group.
  • the unprotected bioorthogonal functional group corresponds to the bioorthogonal functional group described above.
  • a protected bioorthogonal functional group is a group that generates a bioorthogonal functional group upon cleavage of a protecting group. Cleavage of the protecting group can be carried out by specific treatment under conditions (mild conditions) that do not cause protein denaturation or decomposition (eg, cleavage of amide bonds).
  • Such specific treatments include, for example, (a) treatment with one or more substances selected from the group consisting of acidic substances, basic substances, reducing agents, oxidizing agents, and enzymes; (b) treatment with light; treatment with selected physicochemical stimuli, or (c) leaving in the case of using a cleavable linker containing a self-degradable cleavable moiety.
  • Such protecting groups and their cleavage conditions are common general knowledge in the field (e.g., G. Leriche, L. Chisholm, A. Wagner, Bioorganic & Medicinal Chemistry. 20, 571 (2012); Feng P. et al. al ., Journal of American Chemical Society. 132, 1500 (2010).; Bessodes M.
  • reaction conditions eg, reaction temperature, reaction time, reaction solution
  • mild conditions are as described below.
  • protected bioorthogonal functional groups include disulfide residues, ester residues, acetal residues, ketal residues, imine residues, and vicinal diol residues.
  • the bioorthogonal functional group is an unprotected bioorthogonal functional group.
  • the bioorthogonal functional group may be a specific bioorthogonal functional group that has excellent reactivity (eg, reaction level and/or reaction specificity) with other bioorthogonal functional groups.
  • bioorthogonal functional groups include azide residues, alkyne residues (preferably ring groups having a triple bond between carbon atoms, which may be substituted with the above-mentioned substituents), and tetrazine residues. groups, alkene residues, thiol residues, maleimide residues, thiol residues, furan residues, and halocarbonyl residues.
  • Combinations of two bioorthogonal functional groups that can react with each other include, for example, a combination of an azide residue and an alkyne residue, a combination of a tetrazine residue and an alkene residue, a combination of a tetrazine residue and an alkyne residue, and a thiol residue.
  • the functional substance is not particularly limited as long as it is a substance that imparts any function to the antibody, and includes, for example, a drug, a label substance, an affinity substance, a transport substance, and a stabilizer, but preferably a drug or a label. It may be a substance, an affinity substance, or a transport substance.
  • the functional substance may also be a single functional substance or a substance in which two or more functional substances are linked.
  • the drug may be a drug for any disease.
  • diseases include, for example, cancer (e.g., lung cancer, stomach cancer, colon cancer, pancreatic cancer, kidney cancer, liver cancer, thyroid cancer, prostate cancer, bladder cancer, ovarian cancer, uterine cancer, bone cancer, skin cancer, brain tumors, melanoma), autoimmune and inflammatory diseases (e.g. allergic diseases, rheumatoid arthritis, systemic lupus erythematosus), cranial nerve diseases (e.g.
  • the drug may be a prophylactic or therapeutic drug for a disease, or a drug for alleviating side effects.
  • the drug may be an anti-cancer agent.
  • Anticancer agents include, for example, chemotherapeutic agents, toxins, radioactive isotopes, or substances containing them.
  • chemotherapeutic agents include DNA damaging agents, antimetabolites, enzyme inhibitors, DNA intercalating agents, DNA cleaving agents, topoisomerase inhibitors, DNA binding inhibitors, tubulin binding inhibitors, cytotoxic nucleosides, Examples include platinum compounds.
  • toxins include bacterial toxins (eg, diphtheria toxin) and plant toxins (eg, ricin).
  • radioisotopes examples include radioisotopes of hydrogen atoms (e.g., 3 H), radioisotopes of carbon atoms (e.g., 14 C), radioisotopes of phosphorus atoms (e.g., 32 P), and radioisotopes of sulfur atoms.
  • radioisotopes of hydrogen atoms e.g., 3 H
  • radioisotopes of carbon atoms e.g., 14 C
  • radioisotopes of phosphorus atoms e.g., 32 P
  • sulfur atoms examples include radioisotopes of sulfur atoms.
  • Radioactive isotopes e.g., 35 S
  • radioactive isotopes of yttrium e.g., 90 Y
  • radioactive isotopes of technetium e.g., 99m Tc
  • radioactive isotopes of indium e.g., 111 In
  • radioactive isotopes of iodine atoms isotopes e.g., 123 I, 125 I, 129 I, 131 I
  • radioisotopes of samarium e.g., 153 Sm
  • radioisotopes of rhenium e.g., 186 Re
  • radioisotopes of astatine e.g., 211 At
  • radioactive isotopes of bismuth eg, 212 Bi
  • the drugs include auristatin (MMAE, MMAF), maytansine (DM1, DM4), PBD (pyrrolobenzodiazepine), IGN, camptothecin analogs, calicheamicin, duocarmycin, eribulin, anthracycline, dmDNA31, tubulycin. can be mentioned.
  • auristatin MMAE, MMAF
  • maytansine DM1, DM4
  • PBD pyrrolobenzodiazepine
  • IGN camptothecin analogs
  • calicheamicin duocarmycin
  • eribulin eribulin
  • anthracycline dmDNA31
  • tubulycin tubulycin.
  • a labeling substance is a substance that enables detection of a target (eg, tissue, cell, substance).
  • Labeling substances include, for example, enzymes (e.g. peroxidase, alkaline phosphatase, luciferase, ⁇ -galactosidase), affinity substances (e.g. streptavidin, biotin, digoxigenin, aptamer), fluorescent substances (e.g. fluorescein, fluorescein isothiocyanate, rhodamine).
  • luminescent substances e.g., luciferin, aequorin, acridinium esters, tris(2,2'-bipyridyl)ruthenium, luminol
  • radioactive isotopes e.g., those mentioned above
  • examples include substances containing it.
  • An affinity substance is a substance that has an affinity for a target.
  • affinity substances include affinity proteins or peptides such as antibodies, aptamers, lectins, and complementary chains to the target nucleic acid.
  • the affinity substance is preferably an affinity protein or an affinity peptide, and more preferably an antibody.
  • the types of animals from which antibodies used as functional substances are derived are the same as those described above.
  • the type of antibody used as the functional substance may be a polyclonal antibody or a monoclonal antibody.
  • the antibody may also be a divalent antibody (eg, IgG, IgD, IgE), or a quadrivalent or higher antibody (eg, IgA antibody, IgM antibody).
  • Preferably the antibody is a monoclonal antibody.
  • Monoclonal antibodies include, for example, chimeric antibodies, humanized antibodies, human antibodies, antibodies to which predetermined sugar chains have been added (e.g., antibodies modified to have a sugar chain binding consensus sequence such as an N-type sugar chain binding consensus sequence) antibodies), bispecific antibodies, Fc region proteins, Fc fusion proteins, and disulfide bond reduced antibodies.
  • Isotypes of monoclonal antibodies include, for example, IgG (eg, IgG1, IgG2, IgG3, IgG4), IgM, IgA, IgD, IgE, and IgY.
  • antibodies used as functional substances include full-length antibodies and fragments thereof (fragment antibodies). The fragment antibody only needs to maintain binding property to the desired antigen, and includes, for example, Fab, Fab', F(ab') 2 , scFv, and VHH antibodies.
  • the antigenicity of the antibody used as the functional substance may be the same or different from the antigenicity of the immunoglobulin unit in the antibody, antibody derivative, and conjugate of the present invention, and is preferably different.
  • the origin of the antibody used as the functional substance may be the same or different from the origin of the immunoglobulin unit, and is preferably different. Therefore, the antibody used as a functional substance may be a specific chimeric antibody, a specific humanized antibody, or a specific human antibody mentioned in the above-mentioned specific example of a monoclonal antibody, or an antibody derived therefrom. good.
  • the antibody used as the functional substance may also be IgG1, IgG2, IgG3, or IgG4, or an antibody derived therefrom, as mentioned in the specific examples of monoclonal antibodies above.
  • a transport substance is a substance that has the ability to transport a compound.
  • the transport material is preferably a substance that can encapsulate a compound in a protein shell (eg, multimer) (eg, ferritin, virus particles, virus-like particles).
  • a stabilizer is a substance that allows stabilization of antibodies.
  • examples of the stabilizer include diols, glycerin, nonionic surfactants, anionic surfactants, natural surfactants, saccharides, and polyols.
  • the functional substance may also be a peptide, protein, nucleic acid, organic compound, inorganic compound, sugar chain, lipid, high molecular weight polymer, metal (eg, gold), or chelator.
  • peptides include cell membrane-penetrating peptides, blood-brain barrier-penetrating peptides, and peptide drugs.
  • proteins include enzymes, cytokines, fragment antibodies, lectins, interferons, serum albumins, antibodies, and ferritin.
  • nucleic acids include DNA, RNA, and artificial nucleic acids. Nucleic acids also include, for example, RNA interference-inducing nucleic acids (eg, siRNA), aptamers, and antisense.
  • the organic compound include low-molecular organic compounds such as proteolytic chimeric molecules, dyes, and photodegradable compounds.
  • inorganic compounds include silica, talc, and alumina.
  • salts with inorganic acids include salts with hydrogen chloride, hydrogen bromide, phosphoric acid, sulfuric acid, and nitric acid.
  • salts with organic acids include formic acid, acetic acid, trifluoroacetic acid, lactic acid, tartaric acid, fumaric acid, oxalic acid, maleic acid, citric acid, succinic acid, malic acid, benzenesulfonic acid, and p-toluenesulfonic acid. Examples include salt.
  • Salts with inorganic bases include, for example, salts with alkali metals (e.g., sodium, potassium), alkaline earth metals (e.g., calcium, magnesium), and other metals such as zinc, aluminum, and ammonium.
  • Examples of salts with organic bases include salts with trimethylamine, triethylamine, propylene diamine, ethylenediamine, pyridine, ethanolamine, monoalkylethanolamine, dialkylethanolamine, diethanolamine, and triethanolamine.
  • salts with amino acids include salts with basic amino acids (eg, arginine, histidine, lysine, ornithine) and acidic amino acids (eg, aspartic acid, glutamic acid).
  • the salt is preferably a salt with an inorganic acid (eg hydrogen chloride) or a salt with an organic acid (eg trifluoroacetic acid).
  • the present invention provides an affinity substance or a salt thereof comprising first and second affinity portions having an affinity for the constant region in the heavy chain of an antibody.
  • the affinity substance used in the present invention includes first and second affinity moieties that have affinity for the constant region of the heavy chain in the structural unit of an antibody.
  • the building block of an antibody is an immunoglobulin unit containing two heavy chains and optionally two light chains.
  • the building blocks of antibodies are immunoglobulin units that include two heavy chains and two light chains, or two heavy chains and no two light chains.
  • Examples of antibodies containing immunoglobulin units containing two heavy chains and two light chains include divalent antibodies (e.g., IgG, IgD, IgE), or quadrivalent or higher antibodies (e.g., IgA antibodies, IgM antibodies), chimeric antibodies, humanized antibodies, human antibodies, bispecific antibodies, and disulfide bond reduced antibodies.
  • Antibodies that include an immunoglobulin unit that includes two heavy chains and no two light chains include, for example, Fc region proteins, Fc fusion proteins, and disulfide bond reduced antibodies. This is because the Fc region contains a CH2 domain and a CH3 domain as heavy chains, but does not contain a light chain.
  • the first and second affinity moieties may be the same or different. If the first and second affinity moieties are different, the first and second affinity moieties may have affinities for different regions in the constant region of the antibody heavy chain (e.g., the first and second affinity moieties may One of the two affinity moieties has affinity for the CH2 domain and the other has affinity for the CH3 domain), and may also be an affinity substance for the same region in the constant region of the heavy chain of the antibody.
  • the constant region in the heavy chain of an antibody may be a different affinity moiety (e.g., an affinity substance in which both the first and second affinity moieties have affinity for a CH2 domain or a CH3 domain).
  • the different affinity moieties have affinities for the same region within.
  • first and second affinity moieties in order to suppress the association of the first and second affinity moieties with the constant region in a single heavy chain, the first association site of the affinity moiety and the second association site of the second affinity moiety with the constant region of the heavy chain sterically interfere, resulting in a relationship in which one association can suppress the other association. It is preferable that there be. If there is such a relationship, in the antibody constituent unit (an immunoglobulin unit containing two heavy chains and optionally two light chains), two of the affinity substance and the compound containing the reactive group for the antibody will be combined.
  • the first and second affinity moieties include, for example, polymeric substances of predetermined structural units [e.g., peptides (including oligopeptides, polypeptides, and proteins), nucleic acids (including oligonucleic acids and polysaccharides), sugars ( (including oligosaccharides and polysaccharides)] and non-polymeric substances (eg, low molecular weight compounds).
  • the first and second affinity moieties are the same.
  • substances that can be used as the first and second affinity parts that have affinity for the constant region of the heavy chain of an antibody can be obtained by any method.
  • a substance can be selected from a library of arbitrary substances (e.g., a small molecule compound library, a peptide library, an aptamer library, a sugar library, a phage library, an mRNA library, a cDNA library) to obtain a substance that has an affinity for the constant region in the heavy chain of an antibody. It can be obtained by screening for substances having the same properties (eg, high-throughput screening method, phage display method, SELEX method, mRNA display method, ribosome display method, cDNA display method, yeast display method).
  • the Fc region of various antibodies e.g., IgG, IgA, IgM, IgD, IgE
  • a partial peptide present in a specific region e.g, CH1, CH2, CH3
  • the first and second affinity portions can have an affinity for a constant region in the heavy chain of an antibody.
  • the first and second affinity portions may have affinity for the Fc region in the heavy chain of an antibody.
  • the first and second affinity moieties may be used as the constant region of the heavy chain in the CH1 domain, CH2 domain, or CH3 domain, or regions spanning them (e.g., adjacent regions of the CH1 and CH2 domains, the CH2 domain and (adjacent region of the CH3 domain).
  • the first and second affinity moieties may have affinities for the same or different CHX domains (X is 1, 2, or 3), and preferably have affinities for the same domain.
  • “having an affinity for a CHX domain” is not particularly limited as long as it has an affinity for at least a partial region in a CHX domain, and may include an affinity for a partial region in a CHX domain, or an affinity for a CHX domain and other regions. may have affinity for a region spanning the CHX domain (eg, adjacent region). Therefore, the first and second affinity moieties with affinity for the CH2 domain may have affinity for only a partial region in the CH2 domain, or a region spanning the CH2 domain and the CH1 domain or the CH3 domain (e.g., the CH1 domain and the CH2 (adjacent regions of the CH2 domain and CH3 domain).
  • the first and second affinity moieties having affinity for the CH2 domain preferably have affinity for only a partial region in the CH2 domain, or a region spanning the CH2 and CH3 domains (e.g., a region spanning the CH2 and CH3 domains). (adjacent region), or more preferably, only a partial region in the CH2 domain.
  • the first and second affinity portions may be directly linked, or a linker may be provided between the first affinity substance and the second affinity portion. It may be inserted.
  • Reasons such as the binding sites of the first and second affinity parts for the constant region of the antibody heavy chain are close to each other, and the sizes of the first affinity substance and the second affinity part are sufficiently large. Accordingly, the first and second affinity moieties can be linked directly if there is no need to adjust the distance between the first and second affinity moieties.
  • the binding sites of the first and second affinity parts for the constant region of the antibody heavy chain are not close to each other, and the sizes of the first affinity substance and the second affinity part are sufficiently large.
  • a divalent group can be used as a linker.
  • a divalent group may be substituted or unsubstituted. Examples of the divalent group include those mentioned above. Examples of the substituent when the divalent group is substituted include those mentioned above.
  • the linker substances such as peptides, nucleic acids, sugars, other polymeric substances (eg, polyethylene glycol), and divalent hydrocarbon groups (eg, alkyl chains) may be used. Those skilled in the art can appropriately determine the presence or absence of a linker and the type of linker depending on the types of the first and second affinity moieties used.
  • a linker may be inserted from the viewpoint of optimizing the binding of the first and second affinity portions to the constant region of the antibody heavy chain.
  • the constant region in the heavy chain of the antibody to which the first and second affinity portions have affinity may be derived from the animals mentioned above (eg, mammals, birds).
  • the constant region in the heavy chain of the antibody may preferably be a mammalian constant region, more preferably a primate or rodent constant region, and even more preferably a human constant region.
  • the constant region in the heavy chain of an antibody to which the first and second affinity parts have affinity is a divalent antibody (e.g., IgG, IgD, IgE) or a quadrivalent or higher antibody (e.g., IgA antibody, IgM It may be a constant region of an antibody).
  • a constant region is preferably a constant region of a divalent antibody (eg, IgG, IgD, IgE), more preferably an IgG constant region.
  • the first and second affinity moieties may be affinity peptides that have affinity for the constant region in the heavy chain of an antibody.
  • affinity peptides Many peptides have been reported as such affinity peptides.
  • examples of such affinity peptides are: (1) Various IgG-binding peptides (e.g., International Publication No. 2008/054030, International Publication No. 2008/054030, International Publication No. (See Publication No. 2013/027796, International Publication No.
  • PAM Protein A Mimetic peptide that has affinity for a specific region (CH2 domain) of human IgG in general (e.g., Fassina G et al., JOURNAL OF MOLECULAR RECOGNION, 1996, VOL. 6, 564-56 (see 9) ; (3) EPIHRSTLTALL (SEQ ID NO: 27) that has affinity for a specific region (CH2 domain) of human IgG in general (e.g., Ehrlich G.K et al., J. Biochem. Biophys. Methods, 2001, VOL.
  • PAM Protein A Mimetic
  • QSYP SEQ ID NO: 31
  • HWRGWV SEQ ID NO: 32
  • HYFKFD SEQ ID NO: 33
  • HFRRHL SEQ ID NO: 34
  • NARKFYKG SEQ ID NO: 36
  • NKFRGKYK SEQ ID NO: 37
  • Protein A Protein G, protein L, or protein Z, or fragments thereof (e.g., Moks T et al., Eur J Biochem. 1986 May 2; 156 (3): 637-43; Sjobring UJ et al., Biol Chem. 1991 Jan 5; 266 (1): 399-405; Graille Mel al., Structure. 2001 Aug; 9 (8): 679 -87; Nilsson B et al., Protein Eng.
  • IgG-binding peptides that have affinity for specific regions (Fc region or CH2 domain) of human IgG in general (e.g., WO 2018/199337, WO 2019/240287, WO 2019/ 240288, WO 2020/090979).
  • affinity peptide can be obtained by the screening method described above (eg, the method using the library described above, or the display method described above).
  • the 20 types of natural amino acids that constitute proteins or unnatural amino acid residues can be used.
  • the 20 natural amino acids that make up proteins include L-alanine (A), L-asparagine (N), L-cysteine (C), L-glutamine (Q), and L-isoleucine ( I), L-leucine (L), L-methionine (M), L-phenylalanine (F), L-proline (P), L-serine (S), L-threonine (T), L-tryptophan (W) ), L-tyrosine (Y), L-valine (V), L-aspartic acid (D), L-glutamic acid (E), L-arginine (R), L-histidine (H), or L-lysine ( K), and glycine (G) (hereinafter, the notation of L will be omitted).
  • one of the first and second affinity peptides may be an affinity peptide with one lysine residue, and the other may be an affinity peptide without a lysine residue.
  • affinity peptides that have affinity for the constant region of antibody heavy chains and have one lysine residue (e.g., WO 2016/186206, WO 2016/186206, WO 2016/186206, 2018/199337, WO 2019/240287, WO 2019/240288, WO 2020/090979). Therefore, in the present invention, such a peptide can be used as one of the first and second affinity peptides.
  • affinity peptides having affinity for the constant region in the heavy chain of antibodies and having one lysine residue (1) Affinity peptide comprising the amino acid sequence of SEQ ID NOs: 39 to 72 of International Publication No. 2018/199337; (2) an affinity peptide comprising the amino acid sequence of SEQ ID NOs: 5, 6, 37 to 100 of International Publication No. 2019/240288; (3) an affinity peptide comprising the amino acid sequence of SEQ ID NOs: 5, 8 to 57, and 68 to 92 of International Publication No.
  • affinity peptides that have affinity for the constant region in the heavy chain of an antibody and have one lysine residue include, for example, the following (1) to (4): (1) Affinity peptide comprising the amino acid sequence (Fc3K) of RGNCAYHKGQIIWCTYH (SEQ ID NO: 38); (2) In the amino acid sequence of RGNCAYHKGQIIWCTYH (SEQ ID NO: 38), one or two amino acid residues other than lysine residues and cysteine residues are replaced with other amino acid residues other than lysine residues and cysteine residues.
  • an affinity peptide comprising the amino acid sequence as described above and having affinity for the constant region in the heavy chain of an antibody; (3) an affinity peptide containing the amino acid sequence (Z34CK) of FNKQCQRRFYEALHDPNLNEEQRNARIRSIREEC (SEQ ID NO: 39); and (4) an affinity peptide containing the amino acid sequence (Z34CK) of FNKQCQRRFYEALHDPNLNEEQRNARIRSIREEC (SEQ ID NO: 39) other than lysine and cysteine residues.
  • An affinity peptide comprising an amino acid sequence in which the amino acid residue of is replaced with another amino acid residue other than a lysine residue or a cysteine residue, and having an affinity for the constant region of an antibody heavy chain.
  • the two cysteine residues included in the above amino acid sequence may be cross-linked by a disulfide bond.
  • affinity peptides that have affinity for the constant region of antibody heavy chains and do not have lysine residues.
  • the lysine residue in the above-mentioned affinity peptide that has affinity for the constant region in the heavy chain of an antibody and has one lysine residue maintains affinity for the constant region in the heavy chain of the antibody in many cases. Because it is introduced not for the purpose of derivatizing the affinity substance by covalently bonding with other moieties (e.g., partial compounds containing reactive groups) (e.g., International Publication No.
  • an affinity peptide that has affinity for the constant region in the heavy chain of an antibody and does not have a lysine residue has an affinity for the constant region in the heavy chain of an antibody and has one lysine residue.
  • An affinity peptide in which a lysine residue in the affinity peptide is replaced with another amino acid residue preferably a normal natural amino acid residue constituting proteins, other than lysine residues and cysteine residues. Therefore, those having affinity for the constant region in the heavy chain of antibodies can be used.
  • affinity peptide that has affinity for the constant region in the heavy chain of an antibody and does not have a lysine residue: (1) SEQ ID NO: 20-38, 73-75 of International Publication No. 2018/199337 (when Xaa1 is other than a lysine residue), SEQ ID NO: 92; (2) SEQ ID NO: 7, 11-14, 108 of International Publication No. 2019/240288; (3) Among the above (1) to (4) listed as examples of affinity peptides that have affinity for the constant region of the heavy chain of an antibody and have one lysine residue, the lysine residue is different.
  • affinity peptides that do not have a lysine residue among sex peptides.
  • affinity peptides without lysine residues include, for example, the following (5) to (10): (5) an affinity peptide comprising the amino acid sequence (Z34CM) of FNMQCQRRFYEALHDPNLNEEQRNARIRSIREEC (SEQ ID NO: 40); (6) Amino acids in which one or two amino acid residues other than cysteine residues in the amino acid sequence of FNMQCQRRFYEALHDPNLNEEQRNARIRSIREEC (SEQ ID NO: 40) are substituted with lysine residues and other amino acid residues other than cysteine residues.
  • an affinity peptide comprising a sequence and having an affinity for a constant region in an antibody heavy chain; (7) an affinity peptide comprising the amino acid sequence (ProAR) of FNREQQNAFYEILHLPNLNEEQRNGFIQSLRDDPSQSANLLAEA (SEQ ID NO: 41); (8) Amino acids in which one or two amino acid residues other than cysteine residues in the amino acid sequence of FNREQQNAFYEILHLPNLNEEQRNGFIQSLRDDPSQSANLLAEA (SEQ ID NO: 41) are substituted with lysine residues and other amino acid residues other than cysteine residues.
  • an affinity peptide comprising a sequence and having an affinity for a constant region in an antibody heavy chain
  • An affinity peptide comprising the amino acid sequence of RGNCAYHRGQIIWCTYH (SEQ ID NO: 78); and (10) In the amino acid sequence of RGNCAYHRGQIIWCTYH (SEQ ID NO: 78), one or two amino acid residues other than cysteine residues are lysine
  • An affinity peptide comprising an amino acid sequence substituted with another amino acid residue other than a cysteine residue and a cysteine residue, and having an affinity for a constant region in an antibody heavy chain.
  • the two cysteine residues included in the above amino acid sequence may be cross-linked by a disulfide bond.
  • substitution of amino residues may be conservative substitutions.
  • conservative substitution refers to replacing a given amino acid residue with an amino acid residue having a similar side chain.
  • Families of amino acid residues with similar side chains are well known in the art. For example, such families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), and amino acids with uncharged polar side chains.
  • amino acids with nonpolar side chains e.g., glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
  • ⁇ -branched side chains e.g. threonine, valine, isoleucine
  • amino acids with aromatic side chains e.g. tyrosine, phenylalanine, tryptophan, histidine
  • amino acids with side chains containing hydroxyl groups e.g. alcoholic, phenolic
  • amino acids with sulfur-containing side chains eg, cysteine, methionine
  • Amino acids with uncharged polar side chains and amino acids with non-polar side chains are sometimes collectively referred to as neutral amino acids.
  • conservative substitutions of amino acids include substitutions between aspartic acid and glutamic acid, substitutions between arginine and lysine and histidine, substitutions between tryptophan and phenylalanine, substitutions between phenylalanine and valine. , between leucine, isoleucine and alanine, and between glycine and alanine.
  • the affinity substance of the present invention or a salt thereof can be used for the first affinity moiety, even if the first and second affinity peptides are directly linked
  • a linker may be inserted between the peptide and the second affinity peptide, but from the viewpoint of optimizing the binding of the first and second affinity peptides to the constant region of the antibody heavy chain, the linker is inserted between the peptide and the second affinity peptide. Preferably, it is inserted.
  • the linker is preferably a peptide linker.
  • the number of amino acid residues constituting the peptide linker can be appropriately set depending on conditions such as the type of amino acid residue (e.g., ⁇ -amino acid, ⁇ -amino acid, ⁇ -amino acid, preferably ⁇ -amino acid), etc. .
  • a peptide linker may consist of 20 or more amino acid residues.
  • the peptide linker consists of 22 or more, 24 or more, 26 or more, 28 or more, 30 or more, 32 or more, 34 or more, 36 or more, 38 or more, or 40 or more amino acid residues. It may be something.
  • the peptide linker may also consist of less than 50, less than 49, less than 48, less than 47, less than 46, or less than 45 amino acid residues.
  • amino acid residues constituting the peptide linker the above-mentioned natural amino acids or unnatural amino acid residues can be used.
  • the amino acid residues constituting the peptide linker may include only the above-mentioned natural amino acid residues.
  • Amino acid residues suitable for peptide linkers include, but are not limited to, for example, alanine, proline, serine, and glycine.
  • peptide linker those disclosed in WO 2021/112249 and WO 2011/144756 can also be used.
  • the first and second affinity portions are affinity peptides, and a linker is provided between the first affinity peptide and the second affinity peptide. or a salt thereof.
  • an affinity substance has the following formula (A): AP1- LA -AP2 (A) [During the ceremony, AP1 indicates a first affinity peptide with affinity for the constant region in the heavy chain of an antibody; AP2 represents a second affinity peptide with affinity for the constant region in the heavy chain of antibodies; LA indicates a linker. ].
  • first and second affinity peptides represented by AP1 and AP2, respectively are as described above.
  • the linker represented by LA is a divalent group.
  • a divalent group may be substituted or unsubstituted. Examples of the divalent group include those mentioned above. Examples of the substituent when the divalent group is substituted include those mentioned above.
  • materials such as peptides, nucleic acids, sugars, other polymeric substances (eg, polyethylene glycol), divalent hydrocarbon groups (eg, alkyl chains), etc. may be used as linkers.
  • the first and second affinity portions are affinity peptides, and there is a bond between the first affinity peptide and the second affinity peptide. It may also be an affinity polypeptide or a salt thereof containing a peptide linker.
  • Such an affinity polypeptide has the following formula (A'): AP1-PL A -AP2 (A') [During the ceremony, AP1 indicates a first affinity peptide that has affinity for the constant region in the heavy chain of an antibody and is present on the N-terminal side of the affinity polypeptide; AP2 indicates a second affinity peptide that has affinity for the constant region in the heavy chain of an antibody and is present on the C-terminal side of the affinity polypeptide; PLA indicates a peptide linker. ].
  • An affinity substance such as an affinity polypeptide or a salt thereof can contain the above-mentioned natural amino acids or non-natural amino acid residues as constituent amino acid residues.
  • the affinity substance can be used, for example, in a polypeptide expression system using host cells, a cell-free synthesis system, or an organic synthesis system (e.g., solid phase synthesis). It can be manufactured by
  • the affinity substance contains a residue of an unnatural amino acid
  • the affinity substance can be produced, for example, by an organic synthesis system (eg, solid phase synthesis).
  • the affinity substance may contain only natural amino acid residues to enable production of large quantities of the affinity substance by a polypeptide expression system using host cells or a cell-free synthesis system. .
  • the amino group and carboxy group at the terminal of the affinity substance can be protected as appropriate.
  • protecting groups for the N-terminal amino group include alkylcarbonyl groups (acyl groups) (e.g., acetyl groups, propoxy groups, butoxycarbonyl groups such as tert-butoxycarbonyl groups), alkyloxycarbonyl groups (e.g., fluorenyl groups), and alkyloxycarbonyl groups (e.g., fluorenyl groups).
  • the N-terminal amino group may be alkylated, formylated or acetylated.
  • the protecting group for the C-terminal carboxy group include a group capable of forming an ester or an amide.
  • groups capable of forming esters or amides include alkyloxy groups (e.g., methyloxy, ethyloxy, propyloxy, butyloxy, pentyloxy, hexyloxy), aryloxy groups (e.g., phenyloxy, naphthyloxy), aralkyl Examples include oxy groups (eg, benzyloxy) and amino groups.
  • the N-terminal amino acid of the affinity substance is glutamic acid (E) or glutamine (Q)
  • the N-terminus can be protected using those side chains.
  • the N-terminal amino acid is glutamic acid
  • the protected N-terminal glutamic acid can have a cyclic structure of pyroglutamic acid.
  • the N-terminal amino acid is glutamine
  • the protected N-terminal glutamine reacts with the amide group present in the side chain of the N-terminal amino group (NH 2 ) (pyroglutamylation). can have a pyroglutamic acid type cyclic structure. Therefore, the N-terminal amino acid may preferably be glutamic acid or glutamine.
  • the affinity polypeptide may further contain a tripeptide consisting of Gln-Glu-Thr (QET) at the N-terminus.
  • QET Gln-Glu-Thr
  • a signal peptide such as a signal peptide (CspBss) consisting of the amino acid sequence MFNNRIRTAALAGAIAIISTAASGVAIPAFA (SEQ ID NO: 42) can be added to the N-terminal side of QET (Example, International Publication No. 2013/062029, International Publication No. (See issue 2020/090979).
  • CspBss signal peptide
  • the affinity substance of the present invention or a salt thereof can be used, for example, as a synthetic intermediate for the compound of the present invention or a salt thereof containing an affinity substance and an antibody-reactive group. Therefore, in order to easily and uniformly synthesize the compound of the present invention or a salt thereof containing an antibody-reactive group in addition to the affinity substance, the affinity substance of the present invention or its salt has a reactive group for an antibody. may be derivatized to contain only one specific reactive group that enables specific reaction with a moiety containing a compound. When the affinity substance contains only one specific reactive group, both the affinity substance and the reactive group for the antibody can be reacted specifically through the specific reactive group in the affinity substance. Therefore, the compound of the present invention or a salt thereof containing an affinity substance and a reactive group for an antibody can be easily produced as a homogeneous compound.
  • Examples of the above-mentioned specific reactive groups include the following. (1) Amino group (NH 2 , NHR 3 , NR 3 R 4 . R 3 and R 4 are each independently a monovalent group as described above, preferably a monovalent hydrocarbon group, and more (preferably an alkyl group, even more preferably an alkyl group having 1 to 6 carbon atoms); (2) A residue capable of reacting with an amino group.
  • activated ester residues e.g., N-hydroxysuccinimide residues
  • vinyl sulfone residues e.g., vinyl sulfone residues
  • sulfonyl chloride residues isocyanate residues
  • isothiocyanate residues aldehyde residues, 1,4,7,10-tetra Azacyclododecane-1,4,7,10-tetraacetic acid residue, 2-imino-2-methoxyethyl residue, diazonium terephthalic acid residue
  • carboxyl group COOH
  • a residue capable of reacting with a carboxyl group e.g., N-hydroxysuccinimide residues
  • vinyl sulfone residues e.g., vinyl sulfone residues
  • sulfonyl chloride residues e.g., isocyanate residues
  • isothiocyanate residues aldehyde residues
  • an amino group as mentioned above (5) hydroxyl groups (OH) (including alcoholic and phenolic hydroxyl groups); and (6) residues capable of reacting with hydroxyl groups.
  • OH hydroxyl groups
  • residues capable of reacting with hydroxyl groups For example, diazonium residues, diazodicarboxylate residues, 2,3-dihydro-1H-pyrazin-6-one residues.
  • the specific reactive group may preferably be (1) to (4). More preferably, the specific reactive group may be (1) or (2), or (3) or (4). Alternatively, and more preferably, the specific reactive group may be (1) or (3).
  • the specific reactive group is even more preferably (1), particularly preferably an amino group (NH 2 ).
  • the affinity substance or its salt may contain only one amino acid residue containing a specific reactive group.
  • the affinity polypeptide may contain (a) an amino acid residue (e.g., lysine residue, aspartic acid residue) having a specific reactive group (e.g., amino group, carboxyl group, hydroxyl group) in its side chain; , glutamic acid residue, tyrosine residue, threonine residue, or serine residue).
  • the affinity polypeptide contains only one lysine residue having an amino group in its side chain, the N-terminus of the affinity polypeptide is preferably protected (the C-terminus may also be protected).
  • the affinity polypeptide contains only one amino acid residue having a carboxyl group in its side chain (e.g., an aspartic acid residue or a glutamic acid residue), the C-terminus of the affinity polypeptide may be protected. Preferred (the N-terminus may also be protected).
  • the affinity polypeptide does not contain an amino acid residue having an amino group in its side chain (e.g., a lysine residue) and has an amino group at the N-terminus, so that an amino group can be used as a specific reactive group. It may be a polypeptide containing only one. Affinity polypeptides also do not contain amino acid residues having carboxyl groups in their side chains (e.g., aspartic acid residues, glutamic acid residues), and by having a carboxyl group at the C-terminus, they can be used as specific reactive groups. It may be a polypeptide containing only one carboxyl group.
  • the affinity polypeptide containing only one specific reactive group may be a polypeptide containing an amino acid residue having an amino group in its side chain.
  • an affinity polypeptide is produced by an organic synthesis system (e.g., solid-phase synthesis)
  • not only lysine residues which are natural amino acids that constitute proteins, but also other amino acid residues having amino groups in their side chains ( eg, ornithine) can also be used.
  • the affinity polypeptide containing only one specific reactive group can be easily produced not only by an organic synthesis system but also by a polypeptide expression system using host cells and a cell-free synthesis system.
  • the polypeptide may contain only one lysine residue having an amino group (NH 2 ) in its side chain.
  • the lysine residue may be included either in the first affinity peptide, or the second affinity peptide, or in the peptide linker. Lysine residues, if included in the peptide linker, are located proximal to the first affinity peptide or the second affinity peptide (e.g., 10 or less from the first affinity peptide or the second affinity peptide). , 5 or less, or 1 to 3 amino acid residue positions).
  • a lysine residue is included in either the first affinity peptide or the second affinity peptide.
  • the first and second affinity peptides represented by AP1 and AP2, respectively, and the linker represented by LA in the affinity substance represented by the above formula (A) or a salt thereof are the following (1) or It may also have the characteristic (2), preferably the characteristic (1).
  • Affinity substance containing only one amino group as a specific reactive group or a salt thereof (1-1) (i) Affinity in which the first affinity peptide contains only one amino acid residue (preferably a lysine residue) having an amino group in its side chain and has a protected N-terminus (ii) the second affinity peptide is an affinity peptide that does not contain an amino acid residue having an amino group in its side chain, and the linker is a linker that does not contain an amino group; or its salt; (1-2) (i) the first affinity peptide is an affinity peptide that does not contain an amino acid residue having an amino group in its side chain and has an unprotected N-terminus; (ii) the second affinity peptide An affinity substance or a salt thereof, wherein the affinity peptide is an affinity peptide that does not contain an amino acid residue having an amino group in its side chain, and the linker is a linker that does not contain an amino group; (1-3) (i) the first affinity peptide is an affinity peptid
  • the first affinity peptide is an affinity peptide that does not contain an amino acid residue having an amino group in its side chain and has a protected N-terminus;
  • the second affinity peptide An affinity substance or a salt thereof, wherein the affinity peptide is an affinity peptide that does not contain an amino acid residue having an amino group in its side chain, and the linker is a linker containing only one amino group.
  • Affinity substances or salts thereof that contain only one amino group as a specific reactive group contain a group capable of reacting with an amino group (e.g., It may also be an affinity substance or a salt thereof that does not contain a carboxy group.
  • the first affinity peptide may not contain an amino acid residue having a carboxy group in its side chain
  • the second affinity peptide may not contain an amino acid residue having a carboxy group in its side chain and/or may have a protected C-terminus
  • the linker may be a linker that does not contain a carboxy group.
  • the first affinity peptide is an affinity peptide containing only one amino acid residue having a carboxy group in its side chain (preferably an acidic amino acid residue such as aspartic acid or glutamic acid).
  • the second affinity peptide is an affinity peptide that does not contain an amino acid residue having a carboxy group in its side chain, and the linker is a linker that does not contain a carboxy group; salt; (2-2) (i) The first affinity peptide is an affinity peptide that does not contain an amino acid residue having a carboxy group in its side chain, and (ii) the second affinity peptide is an affinity peptide that does not contain an amino acid residue having a carboxy group in its side chain.
  • An affinity peptide that contains only one amino acid residue preferably an acidic amino acid residue such as aspartic acid or glutamic acid
  • the linker does not contain a carboxy group.
  • an affinity substance or a salt thereof that is a linker (2-3) (i) the first affinity peptide is an affinity peptide that does not contain an amino acid residue having a carboxy group in its side chain, and (ii) the second affinity peptide is an affinity peptide that does not contain an amino acid residue having a carboxy group in its side chain; An affinity substance or a salt thereof, which is an affinity peptide that does not contain an amino acid residue in the chain and has an unprotected C-terminus, and the linker is a linker that does not contain a carboxy group; (2-4) (i) the affinity peptide does not contain an amino acid residue having a carboxy group in its side chain, and (ii) the second affinity peptide contains an amino acid residue having a carboxy group in its side chain; and a protected C-terminus, and the linker is a linker containing only one carboxy group, or a salt thereof.
  • an affinity substance or a salt thereof containing only one carboxy group as a specific reactive group may contain a group capable of reacting with a carboxy group (e.g., It may also be an affinity substance or a salt thereof that does not contain an amino group. Therefore, in (2-1) to (2-4) above, the first affinity peptide does not contain an amino acid residue having an amino group in its side chain and/or has a protected N-terminus.
  • the second affinity peptide may be one that does not contain an amino acid residue having an amino group in its side chain, and the linker may be a linker that does not contain an amino group.
  • the first and second affinity peptides represented by AP1 and AP2, respectively, and the peptide linker represented by PL A in the affinity substance represented by the above formula (A') or a salt thereof are the following (1 ') or (2'), preferably (1').
  • Affinity substance containing only one amino group as a specific reactive group or a salt thereof (1-1') (i) An affinity peptide in which the first affinity peptide contains only one amino acid residue (preferably a lysine residue) having an amino group in its side chain and has a protected N-terminus.
  • the second affinity peptide is an affinity peptide that does not contain an amino acid residue having an amino group in its side chain, and the peptide linker contains an amino acid residue having an amino group in its side chain; an affinity substance or a salt thereof that is a peptide linker that does not contain; (1-2') (i) the first affinity peptide is an affinity peptide that does not contain an amino acid residue having an amino group in its side chain and has an unprotected N-terminus, and (ii) the second The affinity peptide is an affinity peptide that does not contain an amino acid residue having an amino group in its side chain, and the peptide linker is a peptide linker that does not contain an amino acid residue having an amino group in its side chain.
  • the first affinity peptide is an affinity peptide that does not contain an amino acid residue having an amino group in its side chain and has a protected N-terminus;
  • the second The affinity peptide is an affinity peptide containing only one amino acid residue (preferably a lysine residue) having an amino group in its side chain, and the peptide linker is an amino acid residue containing an amino group in its side chain.
  • affinity substance or a salt thereof that is a peptide linker that does not contain; (1-4') (i) the first affinity peptide is an affinity peptide that does not contain an amino acid residue having an amino group in its side chain and has a protected N-terminus; (ii) the second affinity peptide
  • the affinity peptide is an affinity peptide that does not contain an amino acid residue having an amino group in its side chain, and the peptide linker is a peptide linker containing only one amino acid residue having an amino group in its side chain, Affinity substance or its salt.
  • Affinity substances or salts thereof that contain only one amino group as a specific reactive group contain a group capable of reacting with an amino group (e.g., It may also be an affinity substance or a salt thereof that does not contain a carboxy group. Therefore, in (1-1') to (1-4') above, the first affinity peptide may not contain an amino acid residue having a carboxy group in its side chain, and the second affinity peptide may not contain an amino acid residue having a carboxy group in its side chain.
  • the peptide linker may not contain an amino acid residue having a carboxy group in its side chain and/or may have a protected C-terminus, and the peptide linker may contain an amino acid residue having a carboxy group in its side chain. It may be a peptide linker that does not contain .
  • Affinity substance containing only one carboxy group as a specific reactive group or a salt thereof (2-1') (i) An affinity peptide in which the first affinity peptide contains only one amino acid residue having a carboxy group in its side chain (preferably an acidic amino acid residue such as aspartic acid or glutamic acid) (ii) the second affinity peptide is an affinity peptide that does not contain an amino acid residue having a carboxy group in its side chain, and the peptide linker contains an amino acid residue having a carboxy group in its side chain; an affinity substance or a salt thereof that is not a peptide linker; (2-2') (i) The first affinity peptide is an affinity peptide that does not contain an amino acid residue having a carboxy group in its side chain, and (ii) the second affinity peptide has a carboxy group in its side chain.
  • affinity peptide that contains only one amino acid residue (preferably an acidic amino acid residue such as aspartic acid or glutamic acid) in its side chain and has a protected C-terminus, and the peptide linker has a carboxy group.
  • An affinity substance or a salt thereof that is a peptide linker that does not contain amino acid residues in its side chain; (2-3') (i) the first affinity peptide is an affinity peptide that does not contain an amino acid residue having a carboxy group in its side chain, and (ii) the second affinity peptide has a carboxy group in its side chain.
  • an affinity substance or a salt thereof containing only one carboxy group as a specific reactive group may contain a group capable of reacting with a carboxy group (e.g., It may also be an affinity substance or a salt thereof that does not contain an amino group. Therefore, in (2-1') to (2-4') above, the first affinity peptide does not contain an amino acid residue having an amino group in its side chain and/or has a protected N-terminus.
  • the second affinity peptide may not contain an amino acid residue having an amino group in its side chain, and the peptide linker may contain an amino acid residue having an amino group in its side chain. A linker that does not include .
  • affinity substance or salt thereof of the present invention is an affinity polypeptide comprising first and second affinity peptides having an affinity for the constant region in the heavy chain of an antibody.
  • an affinity polypeptide can be produced using a host cell containing an expression unit comprising a polynucleotide encoding the affinity polypeptide and a promoter operably linked thereto, or using a cell-free system, etc. It can be prepared.
  • the invention also provides such polynucleotides and host cells, as well as expression vectors that can be used to generate host cells.
  • the polynucleotide of the present invention is a polynucleotide encoding the affinity polypeptide of the present invention.
  • the polynucleotide of the present invention may be DNA or RNA, but DNA is preferred.
  • the host cell of the present invention can be produced, for example, by a method using an expression vector containing the polynucleotide of the present invention (eg, competent cell method, electroporation method), or by genome modification technology.
  • an expression vector containing the polynucleotide of the present invention eg, competent cell method, electroporation method
  • the expression unit can be integrated into the genomic DNA of the host cell by transformation.
  • the expression vector is a non-integrating vector that does not undergo homologous recombination with the genomic DNA of the host cell, the expression unit will not be integrated into the genomic DNA of the host cell through transformation, and will remain in the expression vector within the host cell. It can exist independently from genomic DNA as it is.
  • the expression unit can be incorporated into the genomic DNA of the host cell, and the expression unit inherent in the host cell can be modified. It is possible to do so.
  • genome editing technology e.g., CRISPR/Cas system, Transcription Activator-Like Effector Nucleases (TALEN)
  • TALEN Transcription Activator-Like Effector Nucleases
  • the present invention also provides an expression vector comprising the polynucleotide of the present invention and a promoter operably linked thereto.
  • the expression vectors of the present invention may further contain elements such as terminators, ribosome binding sites, and drug resistance genes that function in host cells. Examples of drug resistance genes include genes resistant to drugs such as tetracycline, ampicillin, kanamycin, hygromycin, and phosphinothricin.
  • the expression vector may also further contain a region that allows homologous recombination with the genome of the host cell for homologous recombination with the genomic DNA of the host cell.
  • an expression vector is designed such that the expression unit it contains is located between a pair of homologous regions (e.g., homology arms homologous to a specific sequence in the host cell's genome, or loxP, or FRT).
  • the genomic region (target of homologous region) of the host cell into which the expression unit is to be introduced is not particularly limited, but may be the locus of a gene that is highly expressed in the host cell.
  • the expression vector may be a plasmid, a viral vector, a phage, or an artificial chromosome. Expression vectors may also be integral or non-integrative vectors.
  • An integrating vector may be a type of vector that is integrated in its entirety into the genome of the host cell. Alternatively, an integrating vector may be a type of vector in which only a portion (eg, an expression unit) is integrated into the genome of the host cell.
  • the expression vector may also be a DNA vector or an RNA vector (eg, a retrovirus).
  • the expression vector may also be a commonly used expression vector.
  • expression vectors examples include pUC (e.g., pUC19, pUC18), pSTV, pBR (e.g., pBR322), pHSG (e.g., pHSG299, pHSG298, pHSG399, pHSG398), RSF (e.g., RSF1010), pACYC ( Examples include pACYC177, pACYC184), pMW (eg, pMW119, pMW118, pMW219, pMW218), pQE (eg, pQE30), and derivatives thereof.
  • pUC e.g., pUC19, pUC18
  • pSTV e.g., pBR322
  • pHSG e.g., pHSG299, pHSG298, pHSG399, pHSG398
  • RSF e.g., RSF101010
  • pACYC Examples include pACYC177, pACY
  • Examples of host cells for expressing the affinity polypeptide of the present invention include bacteria of the genus Escherichia such as Escherichia coli, bacteria of the genus Corynebacterium [e.g., Corynebacterium glutamicum], and various prokaryotic cells including bacteria of the genus Bacillus [e.g., Bacillus subtilis], bacteria of the genus Saccharomyces [e.g., Saccharomyces cerevisiae], bacteria of the genus Pichia [e.g., Pichia sti Pitis (Pichia)
  • Various eukaryotic cells can be used, including Aspergillus stipitis), Aspergillus bacteria (eg, Aspergillus oryzae).
  • insect cells eg, insect cells, plant cells, and animal cells (eg, mammalian cells such as Chinese hamster ovary (CHO) cells) can be used as the host.
  • plant cells eg, mammalian cells such as Chinese hamster ovary (CHO) cells
  • animal cells eg, mammalian cells such as Chinese hamster ovary (CHO) cells
  • host cells include host cells that harbor an expression vector in their cytoplasm and host cells that have a gene of interest introduced onto their genome.
  • affinity polypeptide of the present invention contains a tripeptide consisting of Gln-Glu-Thr (QET) at its N-terminus
  • a polypeptide secretion production method using coryneform bacteria as a host International Publication No. 2013/062029
  • affinity polypeptides that can be prepared. This method can add the N-terminal 3 residues Gln-Glu-Thr (QET) of the Csp mature protein to the N-terminus of the target polypeptide, and also generates polypeptides containing a glutamine residue (Q) at the N-terminus. Since it can be prepared easily and in large quantities, it is suitable for the preparation of affinity polypeptides.
  • various signal peptides such as a signal peptide (CspBss) consisting of the amino acid sequence MFNNRIRTAALAGAIAISTAASGVAIPAFA (SEQ ID NO: 42) can be added to the N-terminal side of QET (Example, International Publication No. 2013/062029, International (See Publication No. 2020/090979).
  • Coryneform bacteria that can be used in this method include, for example, bacteria of the genus Corynebacterium (eg, Corynebacterium glutamicum, Corynebacterium stationis), and bacteria of the genus Brevibacterium.
  • the host cell of the present invention can be cultured, for example, in a medium having the composition described below using a predetermined culture device (eg, test tube, flask, jar fermenter).
  • Culture conditions can be set appropriately.
  • the culture temperature may be 10°C to 37°C
  • the pH may be 6.5 to 7.5
  • the culture time may be 1h to 100h.
  • culture may be performed while controlling the dissolved oxygen concentration.
  • the dissolved oxygen concentration (DO value) in the culture solution may be used as an index for control. Control the aeration and stirring conditions so that the relative dissolved oxygen concentration DO value, when the oxygen concentration in the atmosphere is 21%, does not fall below, for example, 1 to 10%, preferably 3% to 8%. I can do it.
  • the culture may be batch culture or fed-batch culture.
  • culture can be continued by sequentially or continuously adding a solution serving as a sugar source or a solution containing phosphoric acid to the culture solution, either continuously or discontinuously.
  • the promoter for expressing the polynucleotide of the present invention is usually E.
  • Promoters used for heterologous protein production in E. coli can be used, such as PhoA, PhoC, T7 promoter, lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, PL promoter, T5 promoter, etc. Examples include strong promoters, with PhoA, PhoC, and lac being preferred.
  • Vectors include, for example, pUC (e.g., pUC19, pUC18), pSTV, pBR (e.g., pBR322), pHSG (e.g., pHSG299, pHSG298, pHSG399, pHSG398), RSF (e.g., RSF1010), pACYC (e.g., pACYC177, pACYC184), pMW (eg, pMW119, pMW118, pMW219, pMW218), pQE (eg, pQE30), and derivatives thereof, etc. may be used.
  • pUC e.g., pUC19, pUC18
  • pSTV pBR
  • pHSG e.g., pHSG299, pHSG298, pHSG399, pHSG398
  • RSF e.g., RSF101010
  • pACYC e.g., pACY
  • a terminator which is a transcription termination sequence, may be linked downstream of the polynucleotide of the present invention.
  • terminators include T7 terminator, fd phage terminator, T4 terminator, tetracycline resistance gene terminator, and E. coli trpA gene terminator.
  • a medium commonly used for culturing E. coli such as M9-casamino acid medium or LB medium
  • the medium may contain a certain carbon source, nitrogen source, and coenzyme (eg, pyridoxine hydrochloride).
  • coenzyme eg, pyridoxine hydrochloride
  • peptone, yeast extract, NaCl, glucose, MgSO4, ammonium sulfate, potassium dihydrogen phosphate, ferric sulfate, manganese sulfate, etc. may be used.
  • culture conditions and production induction conditions are appropriately selected depending on the marker, promoter, host bacteria, etc. of the vector used.
  • the affinity polypeptide of the present invention can be obtained by disrupting (e.g., sonication, homogenization) or lysing (e.g., lysozyme treatment) the cells. It can be obtained as an object. If the affinity polypeptide is secreted or leaked out of the cells, a sterilizing solution containing the affinity polypeptide can be obtained from the culture solution by centrifugation or membrane filtration.
  • the affinity polypeptide of the present invention can be obtained by subjecting such crushed products, lysates, and disinfectant solutions to techniques such as extraction, precipitation, filtration, and column chromatography.
  • the compound or salt thereof of the present invention has the following properties: (A) an affinity substance comprising first and second affinity parts having an affinity for the constant region in the heavy chain of an antibody; and (B) a reaction with the antibody. Contains sexual groups.
  • affinity substance comprising first and second affinity parts having an affinity for the constant region in the heavy chain of an antibody
  • B a reaction with the antibody.
  • affinity moieties such as affinity peptides, and linkers between affinity moieties such as peptide linkers
  • a reactive group for an antibody As a reactive group for an antibody, a reactive group for an amino acid residue having a side chain that easily reacts among the amino acid residues constituting the antibody (protein) can be used.
  • glycine which has no side chain
  • alanine, isoleucine, leucine, phenylalanine, and valine whose side chains are hydrocarbon groups, are difficult to react to in normal reactions. Inert.
  • the reactive group for antibodies is any one or two of the 14 amino acids consisting of asparagine, glutamine, methionine, proline, serine, threonine, tryptophan, tyrosine, aspartic acid, glutamic acid, arginine, histidine, and lysine. It is a group that can react with the above (eg, 2 types, 3 types, 4 types) of side chains.
  • one or more (e.g., two, three, four) reactive groups may be contained in the compound of the present invention or a salt thereof, Preferably, one type of reactive group may be included in the compound of the invention or a salt thereof.
  • the reactive group for the antibody is a group that can react with the side chain of any one of the above-mentioned 14 types of amino acids constituting the protein.
  • the reactive group for the antibody is more preferably a reactive group specific to the side chain of any one of the amino acids lysine, tyrosine, tryptophan, or cysteine, and still more preferably lysine, tyrosine, or a reactive group specific to the side chain of any one type of amino acid of tryptophan, particularly preferably a reactive group specific to the side chain of lysine or tyrosine, especially the side chain of lysine.
  • WO 2016/186206 WO 2018/199337
  • a reactive group specific to the side chain of a lysine residue is a group that can specifically react with an amino group (NH 2 ) present in the side chain of a lysine residue, such as an activated ester residue.
  • an amino group e.g., N-hydroxysuccinimide residue
  • vinyl sulfone residue e.g., vinyl sulfone residue
  • sulfonyl chloride residue isocyanate residue
  • isothiocyanate residue aldehyde residue, 1,4,7,10-tetraazacyclododecane-1,4 , 7,10-tetraacetic acid residue, 2-imino-2-methoxyethyl residue, diazonium terephthalic acid residue, ⁇ -halogen-substituted acetamide, and ⁇ -halogen-substituted methyl ketone.
  • the reaction between the above-mentioned reactive group specific for the side chain of the lysine residue and the amino group (NH 2 ) present in the side chain of the lysine residue as a linking moiety, for example, Amide residues, urea residues, pyridine residues, carbamate residues, and sulfonamide residues can be generated.
  • a compound of the invention or a salt thereof has the following formula (I): [During the ceremony, R represents a reactive group for antibodies, L represents a linker; A shows an affinity agent that includes first and second affinity moieties that have affinity for the constant region in the heavy chain of an antibody. ].
  • R represents a reactive group for antibodies
  • L represents a linker
  • A shows an affinity agent that includes first and second affinity moieties that have affinity for the constant region in the heavy chain of an antibody.
  • the definitions, examples, and preferred examples of the antibody-reactive group represented by R and the affinity substance represented by A are as described above.
  • a linker is a divalent group.
  • a divalent group may be substituted or unsubstituted. Examples of the divalent group include those mentioned above. Examples of the substituent when the divalent group is substituted include those mentioned above.
  • the compound of the present invention or a salt thereof may further contain a cleavable moiety between the affinity substance and the reactive group for the antibody.
  • the compound represented by the above formula (I) or a salt thereof may contain a linker containing a cleavable moiety.
  • the cleavable moiety is a site that can be cleaved by specific treatment under conditions (mild conditions) that do not cause protein denaturation or decomposition (eg, cleavage of amide bonds). Therefore, the cleavable moiety can be said to be a site (bonds other than amide bonds) that can be cleaved by a specific cleavage treatment under mild conditions.
  • specific treatments include, for example, (a) treatment with one or more substances selected from the group consisting of acidic substances, basic substances, reducing agents, oxidizing agents, and enzymes, (b) physical chemical treatment such as light, etc.
  • reaction conditions eg, reaction temperature, reaction time, reaction solution
  • mild conditions e.g, reaction temperature, reaction time, reaction solution
  • cleavable moieties include disulfide residues, acetal residues, ketal residues, ester residues, carbamoyl residues, alkoxyalkyl residues, imine residues, and tertiary alkyloxycarbamate residues (e.g.
  • tert-butyloxycarbamate residues silane residues, hydrazone-containing residues (e.g., hydrazone residues, acylhydrazone residues, bisarylhydrazone residues), phosphoramidate residues, aconityl residues, trityl residues. groups, azo residues, vicinal diol residues, selenium residues, aromatic ring-containing residues with electron-withdrawing groups, coumarin-containing residues, sulfone-containing residues, unsaturated bond-containing chain residues, and glycosyl residues. Can be mentioned.
  • the aromatic ring group having an electron-withdrawing group is preferably one having an aromatic ring group selected from the group consisting of aryl, aralkyl, an aromatic heterocyclic group, and alkyl having an aromatic heterocyclic group. Alkyl having a cyclic group is more preferred.
  • the electron-withdrawing group is preferably bonded to the 2-position of the ring. Even more preferably, the aromatic ring-containing residue having an electron-withdrawing group is, for example, an aralkyl (eg, benzyl) having an electron-withdrawing group at the 2-position.
  • electron-withdrawing groups include halogen atoms, alkyl substituted with halogen atoms (e.g., trifluoromethyl), boronic acid residues, mesyl, tosyl, triflate, nitro, cyano, phenyl groups, keto groups (e.g., acyl).
  • alkyl i.e., alkylcarbonyl
  • alkoxy i.e., alkyloxy
  • aryl aralkyl, etc. found as prefixes, suffixes, etc. terms in connection with the designation of residues as cleavable moieties.
  • examples, and preferred examples are similar to those described above.
  • ester residues include ordinary ester residues composed of carbon atoms and oxygen atoms (e.g., alkyl esters (e.g., tertiary alkyloxycarbonyl such as tert-butyloxycarbonyl), aryl esters (e.g., phenyloxycarbonyl), syl ester, 2-(diphenylphosphino)benzoate), glycosyl ester residue, orthoester residue], ester residue containing a sulfur atom and an oxygen atom (e.g., ⁇ -thiophenyl ester residue, alkylthioester residue, etc.) (thioester residue), ester residue containing a phosphorus atom and an oxygen atom (eg, phosphodiester residue, phosphotriester residue), and activated ester residue (eg, N-hydroxysuccinimide residue).
  • alkyl esters e.g., tertiary alkyloxycarbonyl such as tert
  • sulfone-containing residues examples include sulfone residues and quinolinylbenzenesulfonate residues.
  • the silane residue is preferably a silane residue having a group selected from the group consisting of alkyl, aryl, aralkyl, and alkoxy.
  • silane residues include dialkyldialkoxysilane residues (eg, dimethyldialkoxysilane, diethyldialkoxysilane), and diaryldialkoxysilane residues (eg, diphenyldialkoxysilane).
  • alkoxyalkyl (i.e., alkyloxyalkyl) residue is a group combining alkyloxy and alkyl as described above, such as a methoxymethyl residue, an ethoxymethyl residue, a methoxyethyl residue, an ethoxyethyl residue.
  • alkyloxyalkyl residue is a group combining alkyloxy and alkyl as described above, such as a methoxymethyl residue, an ethoxymethyl residue, a methoxyethyl residue, an ethoxyethyl residue.
  • An unsaturated bond-containing chain residue is a residue containing an unsaturated bond moiety consisting only of carbon atoms [e.g., vinyl (ethenyl), which is the smallest unit with a double bond between carbon atoms, or a triple bond between carbon atoms.
  • Acetylenyl (ethynyl) which is the smallest unit, or a residue containing an unsaturated bond moiety (e.g., aldehyde, cyano) consisting of a carbon atom and a heteroatom (e.g., nitrogen atom, sulfur atom, oxygen atom).
  • unsaturated bond-containing chain residues include vinyl ether residues, cyanoethyl residues, ethylene residues, and malondialdehyde residues.
  • acidic substances include inorganic acidic substances such as hydrochloric acid, sulfuric acid, and nitric acid, as well as formic acid, acetic acid, 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid, and 3- Examples include organic acidic substances such as morpholinopropanesulfonic acid, sodium dihydrogen phosphate, citric acid, dodecyl sulfuric acid, N-dodecanoylsarcosinate, and trifluoroacetic acid.
  • Sites that can be cleaved by acidic substances include, for example, alkyloxyarylalkyl residues, tertiary alkyloxycarbamate residues, acetal residues, silane residues, imine residues, vinyl ether residues, and ⁇ -thiopropionate residues. group, trityl residue, hydrazone residue, aconityl residue, orthoester residue, carbamoyl residue, and 2-(diphenylphosphino)benzoate residue.
  • Examples of basic substances include inorganic basic substances such as sodium hydroxide, potassium hydroxide, sodium acetate, potassium acetate, ammonium acetate, and hydroxylamine, triethylamine, N,N' -Organic basic substances such as diisopropylamine.
  • Sites that can be cleaved by basic substances include, for example, silane residues, cyanoethyl residues, sulfone residues, ethylene residues, glycosyldisuccinate residues, ⁇ -thiophenyl ester residues, and unsaturated vinyl sulfide residues. , malondialdehyde residue, acylhydrazone residue, and alkylthioester residue.
  • reducing agent examples include cysteine, dithiothreitol, reduced glutathione, and ⁇ -mercaptoethanol.
  • sites that can be cleaved with a reducing agent include disulfide residues, alkoxyalkyl residues, and azo residues.
  • oxidizing agent examples include sodium periodate and oxidized glutathione.
  • sites that can be cleaved by an oxidizing agent include vicinal diol residues and selenium residues.
  • Examples of the enzyme include trypsin, papain, TEV, thrombin, cathepsin B, cathepsin D, cathepsin K, caspase, protease, matrix metalloprotease, lipase, endoglycosidase, and PN gauze F.
  • Examples of enzymatically cleavable sites include ester residues, phosphodiester residues, and glycosyl residues.
  • photo-cleavable sites examples include 2-nitrobenzyl residues, phenacyl ester residues, 8-quinolinebenzenesulfonate residues, coumarin residues, phosphotriester residues, bisarylhydrazone residues, and bimanyl hydrazone residues.
  • Examples include dithiopropionic acid residues.
  • self-degradable cleavable moiety examples include activated ester residues (eg, N-hydroxysuccinimide residues).
  • the cleavable moiety may be one that can generate a bioorthogonal functional group on the reactive group side by cleavage.
  • cleavable moieties include, for example, disulfide residues, ester residues (including conventional ester residues, and other ester residues mentioned above such as thioester residues), acetal residues (conventional ester residues), and other acetal residues such as thioacetal residues), ketal residues, imine residues, and vicinal diol residues.
  • the compound of the present invention or a salt thereof contains a cleavable moiety that can generate a bioorthogonal functional group on the reactive group side by cleavage
  • the compound has the following formula (Ia): [During the ceremony, R represents a reactive group for antibodies; L 1 indicates the first linker, L2 represents a second linker; CLE (B) indicates a cleavable moiety that can generate a bioorthogonal functional group on the reactive group side by cleavage, A shows an affinity agent that includes first and second affinity moieties that have affinity for the constant region in the heavy chain of an antibody. ].
  • the first linker represented by L 1 and the second linker represented by L 2 may be the same or different divalent groups.
  • a divalent group may be substituted or unsubstituted. Examples of the divalent group include those mentioned above. Examples of the substituent when the divalent group is substituted include those mentioned above.
  • the total number of atoms constituting the main chains in the first linker and the second linker may be 2 to 10.
  • the total number of such atoms may be 3 or more, or 4 or more.
  • the total number of such atoms may be 9 or less, 8 or less, or 7 or less. More specifically, the total number of such atoms may be 3-9, 4-8, or 4-7.
  • the number of atoms constituting the main chains of the first linker and the second linker may be 1 to 9, respectively.
  • the number of such atoms may be 2 or more, or 3 or more.
  • the number of such atoms may be 8 or less, 7 or less, or 6 or less. More specifically, the number of such atoms may be 2-8, 3-7, or 3-6.
  • the main chains in the first linker and the second linker are composed of a chain structure, a cyclic structure, or a structure containing a combination thereof.
  • the number of atoms in the main chain can be determined by counting the number of atoms in the chain structure.
  • the main chain has a structure including a cyclic structure, it can be determined by counting the predetermined number of atoms constituting the cyclic structure as the number of atoms in the main chain.
  • the number of atoms in the main chain in a cyclic structure can be determined by counting the number of atoms in the shortest path connecting two bonds in the cyclic structure (for example, the following (a) to (d) ).
  • the number of atoms in the main chain is the number of atoms in the chain structure that does not include a cyclic structure, and the number of atoms that connect two bonds in the cyclic structure. It can be determined by adding up the number of atoms in the shortest path.
  • the method of counting the number of atoms in the main chain is the same for other linkers. ⁇ is a bond.
  • the compound of the present invention or a salt thereof has the following formula (Ia-1): [During the ceremony, X represents a leaving group, W 1 , W 2 and W 3 each independently represent an oxygen atom or a sulfur atom, L3 indicates a third linker, L 4 indicates the fourth linker, S represents a sulfur atom, A shows an affinity agent that includes first and second affinity moieties that have affinity for the constant region in the heavy chain of an antibody. ] or a salt thereof.
  • the definition, examples, and preferred examples of the affinity substance represented by A are as described above.
  • the leaving group represented by X is a group that can be left off by reaction between the carbon atom in C ⁇ W 1 adjacent to X and the amino group.
  • Such leaving groups include, for example: (a) R A -S (where R A is a hydrogen atom, a monovalent hydrocarbon group that may have a substituent, or a monovalent heterocyclic group that may have a substituent) and S represents a sulfur atom); (b) R A -O (where R A is a hydrogen atom, a monovalent hydrocarbon group that may have a substituent, or a monovalent heterocyclic group that may have a substituent) and O represents an oxygen atom); (c) R A -(R B -)N (where R A and R B are each independently a hydrogen atom, a monovalent hydrocarbon group that may have a substituent, or a substituent or (d) a halogen atom.
  • the leaving group represented by X may be: (a) R A -S (where R A is a hydrogen atom, a monovalent hydrocarbon group that may have a substituent, or a monovalent heterocyclic group that may have a substituent) and S represents a sulfur atom); (b) R A -O (where R A is a hydrogen atom, a monovalent hydrocarbon group that may have a substituent, or a monovalent heterocyclic group that may have a substituent) or (c) R A - (R B -) N (where R A and R B each independently have a hydrogen atom or a substituent; (N represents a nitrogen atom.)
  • the leaving group represented by X may be: (a) R A -S (where R A is a hydrogen atom, a monovalent hydrocarbon group that may have a substituent, or a monovalent heterocyclic group that may have a substituent) or (b) R A -O (where R A is a hydrogen atom, a monovalent hydrocarbon group that may have a substituent, or a substituted represents a monovalent heterocyclic group which may have a group, and O represents an oxygen atom).
  • the leaving group represented by X may be: (a) R A -S (where R A is a hydrogen atom, a monovalent hydrocarbon group that may have a substituent, or a monovalent heterocyclic group that may have a substituent) and S represents a sulfur atom).
  • the leaving group denoted by X may be: (a') R A -S (Here, R A represents a monovalent aromatic hydrocarbon group (e.g., phenyl) that may have a substituent, and S represents a sulfur atom.) .
  • W 1 , W 2 and W 3 each independently represent an oxygen atom or a sulfur atom.
  • W 1 , W 2 and W 3 may be oxygen atoms.
  • the third linker represented by L 3 and the fourth linker represented by L 4 may be the same or different divalent groups.
  • a divalent group may be substituted or unsubstituted. Examples of the divalent group include those mentioned above. Examples of the substituent when the divalent group is substituted include those mentioned above.
  • the total number of atoms forming the main chains in the third linker and the fourth linker may be 2 to 10.
  • the total number of such atoms may be 3 or more, or 4 or more.
  • the total number of such atoms may be 9 or less, 8 or less, or 7 or less. More specifically, the total number of such atoms may be 3-9, 4-8, or 4-7.
  • the number of atoms constituting the main chains of the third linker and the fourth linker may be 1 to 9, respectively.
  • the number of such atoms may be 2 or more, or 3 or more.
  • the number of such atoms may be 8 or less, 7 or less, or 6 or less. More specifically, the number of such atoms may be 2-8, 3-7, or 3-6.
  • the compound of the present invention or a salt thereof may further contain a bioorthogonal functional group between the antibody-reactive group and the cleavable moiety.
  • the bioorthogonal functional group is as described above.
  • the bioorthogonal functional group is an azide residue, an alkyne residue (preferably a ring group having a triple bond between carbon atoms, which may be substituted with a substituent as described above), or a tetrazine residue. , alkene residues, thiol residues, maleimide residues, thiol residues, furan residues, and halocarbonyl residues.
  • the fifth linker represented by L 5 and the sixth linker represented by L 6 may be the same or different divalent groups.
  • a divalent group may be substituted or unsubstituted. Examples of the divalent group include those mentioned above. Examples of the substituent when the divalent group is substituted include those mentioned above.
  • the total number of atoms forming the main chains in the fifth linker and the sixth linker may be 2 to 10.
  • the total number of such atoms may be 3 or more, or 4 or more.
  • the total number of such atoms may be 9 or less, 8 or less, or 7 or less. More specifically, the total number of such atoms may be 3-9, 4-8, or 4-7.
  • the number of atoms constituting the main chains of the fifth linker and the sixth linker may be 1 to 9, respectively.
  • the number of such atoms may be 2 or more, or 3 or more.
  • the number of such atoms may be 8 or less, 7 or less, or 6 or less. More specifically, the number of such atoms may be 2-8, 3-7, or 3-6.
  • the group containing a bioorthogonal functional group represented by B may be a group consisting of a bioorthogonal functional group, or may be a group containing a bioorthogonal functional group and another moiety.
  • Other portions include, for example, a connecting portion between a bioorthogonal functional group and a linker.
  • the connecting portion is, for example, a divalent group.
  • a divalent group may be substituted or unsubstituted.
  • the compound of the present invention or a salt thereof has the following formula (Ib-1): [During the ceremony, X represents a leaving group, W 1 , W 2 and W 3 each independently represent an oxygen atom or a sulfur atom, L 7 indicates the seventh linker, L8 indicates the eighth linker, B represents a group containing a bioorthogonal functional group, V represents an oxygen atom or a sulfur atom, A shows an affinity agent that includes first and second affinity moieties that have affinity for the constant region in the heavy chain of an antibody. ] or a salt thereof.
  • the definitions, examples, and preferred examples of the leaving group represented by X, the group containing a bioorthogonal functional group represented by B, and the affinity substance represented by A are as described above.
  • W 1 , W 2 and W 3 each independently represent an oxygen atom or a sulfur atom.
  • W 1 , W 2 and W 3 may be oxygen atoms.
  • the seventh linker represented by L 7 and the eighth linker represented by L 8 may be the same or different divalent groups.
  • a divalent group may be substituted or unsubstituted. Examples of the divalent group include those mentioned above. Examples of the substituent when the divalent group is substituted include those mentioned above.
  • the total number of atoms forming the main chains in the seventh linker and the eighth linker may be 2 to 10.
  • the total number of such atoms may be 3 or more, or 4 or more.
  • the total number of such atoms may be 9 or less, 8 or less, or 7 or less. More specifically, the total number of such atoms may be 3-9, 4-8, or 4-7.
  • the number of atoms constituting the main chains of the seventh linker and the eighth linker may be 1 to 9, respectively.
  • the number of such atoms may be 2 or more, or 3 or more.
  • the number of such atoms may be 8 or less, 7 or less, or 6 or less. More specifically, the number of such atoms may be 2-8, 3-7, or 3-6.
  • V represents an oxygen atom or a sulfur atom.
  • V may be a sulfur atom.
  • the compound of the present invention or a salt thereof can easily modify only one heavy chain in the structural unit of an antibody.
  • the compound of the present invention or a salt thereof can also easily modify only one heavy chain in the antibody structural unit and provide a regioselectively modified antibody.
  • the above-mentioned series of compounds or salts thereof can be produced by reacting the affinity substance of the present invention with a partial compound containing a group reactive with antibodies.
  • a suitable organic solvent system e.g., an organic solvent containing an alkyl halide (e.g., methyl halide), such as CH 2 Cl 2 , and an amine, such as triethylamine
  • the reaction time is, for example, 1 minute to 20 hours, preferably 10 minutes to 15 hours, more preferably 20 minutes to 10 hours, and even more preferably 30 minutes to 8 hours.
  • affinity substance-modified antibody or salt thereof 4-1 An affinity substance-modified antibody or a salt thereof comprising at least one affinity substance (comprising a first and a second affinity moiety) An affinity substance-modified antibody or a salt thereof is provided, which comprises an affinity substance comprising an affinity portion of the present invention in the constant region of the heavy chain of the antibody.
  • affinity substances, antibodies, and their constituent elements e.g., affinity portions such as affinity peptides, linkers between affinity portions such as peptide linkers, and constant regions
  • an affinity substance-modified antibody or a salt thereof comprises (a) a constitutional unit of an antibody (an immunoglobulin unit comprising two heavy chains and optionally two light chains), and (b) an affinity substance. and (c) the affinity substance is introduced only into the constant region of one heavy chain in the immunoglobulin unit (i.e., the affinity substance is introduced into the constant region of one heavy chain in the immunoglobulin unit).
  • a modified antibody may be introduced into the constant region of the other heavy chain, and an affinity substance-modified antibody may not be introduced into the constant region of the other heavy chain.
  • Affinity substance-modified antibodies or salts thereof include asparagine, glutamine, methionine, proline, serine, threonine, tryptophan, tyrosine, aspartic acid, glutamic acid, arginine, and histidine present in the constant region (preferably Fc region or CH2 domain). , and lysine through the modification of the functional group in the side chain of any one or two or more (e.g., 2, 3, 4) of the 14 amino acid residues that contain an affinity substance. be able to.
  • the affinity substance-modified antibody or its salt preferably has a functional group in the side chain of any one of lysine, tyrosine, tryptophan, or cysteine present in the constant region (preferably Fc region or CH2 domain). via modification of groups, more preferably via modification of functional groups in the side chains of any one of the amino acids lysine, tyrosine or tryptophan, even more preferably in the side chains of lysine or tyrosine.
  • Affinity substances can be included through modification of functional groups, particularly preferably through modification of amino groups in the side chains of lysine. The positions of these amino acid residues in the constant region are as described above.
  • the modification position of an antibody or its salt by an affinity substance can be confirmed by peptide mapping.
  • Modifications may be regioselective, as described above. Therefore, in formulas (II), (IIa), (IIa-1), (IIb), and (IIb-1) described below, the immunoglobulin unit is , may have a corresponding modification unit regioselectively.
  • the affinity substance-modified antibody comprises a constant region (preferably an Fc region) of one heavy chain in an antibody structural unit (an immunoglobulin unit comprising two heavy chains and optionally two light chains). or CH2 domain) through modification of the amino group in the side chain of one or more (preferably 1 or 2, more preferably 1) lysine residues (in other words, , containing the affinity substance through the amino group in the side chain of a lysine residue in the constant region of one heavy chain in the immunoglobulin unit and in the side chain of a lysine residue in the constant region of the other heavy chain. (does not contain affinity substances via amino groups).
  • the position of one or more (preferably 1 or 2, more preferably 1) lysine residues is at position 246/248, 288/290, or 317 of the human IgG heavy chain according to EU numbering.
  • Modifications may be regioselective, as described above. Therefore, in formulas (II), (IIa), (IIa-1), (IIb), and (IIb-1) described below, the immunoglobulin unit is , may have a corresponding modification unit regioselectively.
  • affinity substance-modified antibodies or salts thereof involves reacting a compound of the present invention or a salt thereof with an antibody or a salt thereof containing an immunoglobulin unit comprising two heavy chains and optionally two light chains. This can be done by
  • the equivalent amount of the compound of the present invention or its salt relative to the antibody in the reaction varies depending on factors such as the compound of the present invention or its salt and the type of antibody, so particularly Although not limited, it is, for example, from 1 to 100, preferably from 2 to 80, more preferably from 4 to 60, even more preferably from 5 to 40, particularly preferably from 6 to 20.
  • Such a reaction can be appropriately carried out under conditions (mild conditions) that do not cause protein denaturation or decomposition (eg, cleavage of amide bonds).
  • a suitable reaction system such as a buffer.
  • the pH of the buffer solution is, for example, 5 to 9, preferably 5.5 to 8.5, and more preferably 6.0 to 8.0.
  • the buffer may also contain a suitable catalyst.
  • the reaction time is, for example, 1 minute to 20 hours, preferably 10 minutes to 15 hours, more preferably 20 minutes to 10 hours, and even more preferably 30 minutes to 8 hours.
  • the affinity substance-modified antibody or salt thereof has the following formula (II): [During the ceremony, Ig refers to an immunoglobulin unit containing two heavy chains and optionally two light chains; L represents a linker; A indicates an affinity substance comprising first and second affinity moieties having affinity for the constant region in the heavy chain of an antibody; The average modification percentage r of the immunoglobulin units by the affinity substance is 65-135%. ] or a salt thereof may be used.
  • the average modification percentage r of the immunoglobulin unit by the affinity substance is 65-135%.
  • the average modification percentage r is 66% or more, 67% or more, 68% or more, 69% or more, 70% or more, 72% or more, 74% or more, 76% or more, 78% or more, 80% or more, 82% or more, It may be 84% or more, 86% or more, 88% or more, 90% or more, 92% or more, 94% or more, or 96% or more.
  • the average modification percentage r is also 130% or less, 125% or less, 120% or less, 115% or less, 110% or less, 105% or less, 100% or less, 98% or less, 96% or less, 94% or less, 92% or less , 90% or less, 88% or less, 86% or less, 84% or less, 82% or less, or 80% or less.
  • the average modification percentage r can be determined by mass spectrometry (DAR calculator (Agilent software) can be used in combination; see Examples).
  • the average modification percentage r is also preferably 65-100%, more preferably 70-100%, even more preferably 75-100%, particularly preferably 80-100%, 85-100%, 90-100%, or It may be 95-100%.
  • the upper limit may be a value below the above-mentioned percentages, such as 98% or below, or 96% or below.
  • the average modification percentage r may be 96-100%, 97-100%, 98-100%, 99-100%, or 100%.
  • the degree of the average modification percentage r described above can be similarly applied to other average modification percentages r. That is, the degree of the above-mentioned average modification percentage r depends not only on the below-mentioned average modification percentage r due to the affinity substance, but also on the below-mentioned average modification percentage r due to any modification (e.g., bioorthogonal functional group, functional substance). can be applied as well.
  • a compound represented by formula (I) or a salt thereof is combined with two heavy chains and optionally two light chains. This can be carried out by reacting with an antibody containing an immunoglobulin unit or a salt thereof.
  • the affinity substance-modified antibody or a salt thereof may further include a cleavable moiety between the affinity substance and the antibody (immunoglobulin unit).
  • the antibody or salt thereof containing the structural unit represented by the above formula (II) may contain a linker containing a cleavable portion. Definitions, examples, and preferred examples of cleavable moieties are as described above.
  • the cleavable moiety may be one that can generate a bioorthogonal functional group on the antibody (immunoglobulin unit) side by cleavage.
  • cleavable moieties include, for example, disulfide residues, ester residues (including conventional ester residues, and other ester residues mentioned above such as thioester residues), acetal residues (conventional ester residues), and other acetal residues such as thioacetal residues), ketal residues, imine residues, and vicinal diol residues.
  • the affinity substance-modified antibody or its salt contains a cleavable moiety that can generate a bioorthogonal functional group on the antibody (immunoglobulin unit) side by cleavage
  • Ig refers to an immunoglobulin unit containing two heavy chains and optionally two light chains
  • L 1 indicates the first linker
  • L2 represents a second linker
  • CLE (B) represents a cleavable moiety that can generate a bioorthogonal functional group on the immunoglobulin unit side by cleavage
  • A indicates an affinity substance comprising first and second affinity moieties having affinity for the constant region in the heavy chain of an antibody
  • the average modification percentage r of the immunoglobulin units by the affinity substance is 65-135%.
  • an immunoglobulin unit denoted Ig an immunoglobulin unit denoted Ig
  • a first linker denoted L1 a second linker denoted L2
  • an affinity substance denoted A an affinity substance denoted A
  • an r denoted The average modification percentages, as well as antibody definitions, examples, and preferred examples are as described above.
  • a compound represented by formula (Ia) or a salt thereof is combined with two heavy chains and optionally two light chains. This can be carried out by reacting with an antibody containing an immunoglobulin unit or a salt thereof.
  • the affinity substance-modified antibody or salt thereof has the following formula (IIa-1): [During the ceremony, Ig refers to an immunoglobulin unit containing two heavy chains and optionally two light chains; W 1 , W 2 and W 3 each independently represent an oxygen atom or a sulfur atom, L 3 indicates a third linker, L 4 indicates the fourth linker, S represents a sulfur atom, A indicates an affinity substance comprising first and second affinity moieties having affinity for the constant region in the heavy chain of an antibody; The average modification percentage r of the immunoglobulin units by the affinity substance is 65-135%. ] or a salt thereof may be used.
  • a compound represented by formula (Ia-1) or a salt thereof is combined with two heavy chains and, if necessary, two heavy chains. This can be carried out by reacting with an antibody containing an immunoglobulin unit containing a light chain or a salt thereof.
  • the affinity substance-modified antibody or its salt may further contain a bioorthogonal functional group between the antibody (immunoglobulin unit) and the cleavable moiety.
  • the bioorthogonal functional group is as described above.
  • the bioorthogonal functional group is an azide residue, an alkyne residue (preferably a ring group having a triple bond between carbon atoms, which may be substituted with a substituent as described above), or a tetrazine residue. , alkene residues, thiol residues, maleimide residues, thiol residues, furan residues, and halocarbonyl residues.
  • the affinity substance-modified antibody or its salt further contains a bioorthogonal functional group between the antibody (immunoglobulin unit) and the cleavable moiety
  • formula (IIb) [During the ceremony, Ig refers to an immunoglobulin unit containing two heavy chains and optionally two light chains; L 5 indicates the fifth linker, L 6 indicates the sixth linker, B represents a group containing a bioorthogonal functional group, CLE indicates a cleavable moiety; A indicates an affinity substance comprising first and second affinity moieties having affinity for the constant region in the heavy chain of an antibody; The average modification percentage r of the immunoglobulin units by the affinity substance is 65-135%. ] or a salt thereof may be used.
  • An immunoglobulin unit denoted by Ig a fifth linker denoted by L5 , a sixth linker denoted by L6 , a group containing a bioorthogonal functional group denoted by B, a cleavable site denoted by CLE, a cleavage site denoted by A.
  • the affinity substances represented, and the average modification percentage represented by r, as well as the definitions, examples, and preferred examples of antibodies are as described above.
  • a compound represented by formula (Ib) or a salt thereof is combined with two heavy chains and optionally two light chains. This can be carried out by reacting with an antibody containing an immunoglobulin unit or a salt thereof.
  • the affinity substance-modified antibody or salt thereof has the following formula (IIb-1): [During the ceremony, Ig refers to an immunoglobulin unit containing two heavy chains and optionally two light chains; W 1 , W 2 and W 3 each independently represent an oxygen atom or a sulfur atom, L 7 indicates the seventh linker, L8 indicates the eighth linker, B represents a group containing a bioorthogonal functional group, V represents an oxygen atom or a sulfur atom; A represents an affinity substance comprising first and second affinity moieties having an affinity for the constant region in the heavy chain of an antibody; The modification percentage r of the immunoglobulin unit by the affinity substance is 65-135%. ] or a salt thereof may be used.
  • It contains an immunoglobulin unit designated Ig, atoms designated W 1 , W 2 and W 3 , a seventh linker designated L 7 , an eighth linker designated L 8 , and a bioorthogonal functional group designated B.
  • the group, the atom denoted by V, the affinity substance denoted by A, and the average modification percentage denoted by r, as well as definitions, examples, and preferred examples of antibodies are as described above.
  • a compound represented by formula (Ib-1) or a salt thereof is combined with two heavy chains and optionally two This can be carried out by reacting with an antibody containing an immunoglobulin unit containing a light chain or a salt thereof.
  • the affinity substance-modified antibody or its salt may further contain an additional modification moiety.
  • an affinity substance-modified antibody or a salt thereof can be modified to further include an additional modification moiety. Additional modifying moieties may be introduced into the heavy chain or light chain of the antibody, preferably in the heavy chain of the antibody (particularly in the constant region of the heavy chain).
  • the additional modifying moiety may be an additional affinity agent that includes a third affinity moiety that has affinity for the constant region in the heavy chain of an antibody.
  • the "affinity moiety" in the “third affinity moiety” and the “affinity substance” in the “additional affinity substance” are each the same as described above.
  • the third affinity moiety may be the same or different from the first and/or second affinity moieties described above, but is preferably different.
  • an additional modifying moiety comprising a third affinity moiety having affinity for a constant region in a heavy chain of an antibody is present at one or more positions in the constant region of said two heavy chains. It may be introduced into the constant regions of the two heavy chains through modification of the amino groups in the side chains of lysine residues.
  • An affinity substance-modified antibody or a salt thereof is produced by combining two heavy chain constant regions (preferably Fc Additional modification moieties can be included through modification of the amino group in the side chain of one or more (preferably one or two, more preferably one) lysine residues in the CH2 domain or CH2 domain).
  • the position of one or more (preferably 1 or 2, more preferably 1) lysine residues is at position 246/248, 288/290, or 317 of the human IgG heavy chain according to EU numbering.
  • the position at which the additional modification moiety is introduced including the third affinity moiety that has affinity for the constant region in the heavy chain of the antibody, is located between the first and second affinity moieties that have affinity for the constant region in the heavy chain of the antibody.
  • the position is preferably different from the position at which the affinity substance containing the sexual moiety is introduced.
  • the additional modification moieties are preferably introduced into lysine residues at positions 288/290 or 317.
  • lysine residues at positions 288/290 are more preferred.
  • the position where the affinity substance is introduced is a lysine residue at position 288/290
  • the position where the additional modification moiety is introduced is preferably a lysine residue at position 246/248 or 317; A lysine residue at position /248 is more preferred.
  • the affinity substance is introduced to the lysine residue at position 317
  • the additional modification moiety is preferably introduced to the lysine residue at positions 246/248 or 288/290.
  • Affinity substance-modified antibodies or salts thereof comprising at least two affinity substances (including first, second, third and fourth affinity moieties) a first modification moiety comprising a first affinity substance comprising first and second affinity moieties having an affinity for a constant region in the heavy chain of an antibody; and third and fourth affinity moieties having an affinity for a constant region in the heavy chain of an antibody
  • the present invention provides an affinity substance-modified antibody or a salt thereof comprising first and second modifying moieties, the second modifying moiety comprising a second affinity substance comprising the first and second modifying moieties in the constant region of the heavy chain of the antibody.
  • affinity substances e.g., affinity portions such as affinity peptides, linkers between affinity portions such as peptide linkers, and constant regions
  • affinity portions e.g., affinity peptides, linkers between affinity portions such as peptide linkers, and constant regions
  • the first and second modifying moieties may be the same or different, and are preferably different.
  • such an affinity substance-modified antibody or a salt thereof comprises (a) a constitutional unit of an antibody (an immunoglobulin unit comprising two heavy chains and optionally two light chains); and (b) (c) the first modification moiety is introduced into the constant region of the first heavy chain in the immunoglobulin unit; and (d) the second modification moiety comprises the first and second modification moieties.
  • the modified moiety may be introduced into the constant region of the second heavy chain in the immunoglobulin unit.
  • Affinity substance-modified antibodies or salts thereof include asparagine, glutamine, methionine, proline, serine, threonine, tryptophan, tyrosine, aspartic acid, glutamic acid, arginine, and histidine present in the constant region (preferably Fc region or CH2 domain).
  • the first affinity A first modification moiety comprising a substance and a second modification moiety comprising a second affinity substance can be included.
  • the affinity substance-modified antibody or its salt preferably has a functional group in the side chain of any one of lysine, tyrosine, tryptophan, or cysteine present in the constant region (preferably Fc region or CH2 domain).
  • the immunoglobulin unit may have a corresponding modification unit regioselectively via the functional group in the side chain of the amino acid residue.
  • the affinity substance-modified antibody comprises a constant region of the first heavy chain (preferably an Fc the first affinity substance comprising said first affinity substance through modification of the amino group in the side chain of one or more (preferably 1 or 2, more preferably 1) lysine residues in the and the side chains of one or more (preferably 1 or 2, more preferably 1) lysine residues in the constant region (preferably the Fc region or CH2 domain) of the second heavy chain.
  • a second modification moiety containing the second affinity substance can be included through modification of the amino group therein. More specifically, the position of one or more (preferably 1 or 2, more preferably 1) lysine residues is at position 246/248, 288/290, or 317 of the human IgG heavy chain according to EU numbering. There may be.
  • Modifications may be regioselective, as described above. Therefore, in the below-mentioned formula, the immunoglobulin unit may have a corresponding modification unit regioselectively via the amino group in the side chain of the lysine residue.
  • affinity substance-modified antibodies or salts thereof can be carried out as follows: (1) The compound of the present invention or a salt thereof is reacted with an antibody containing an immunoglobulin unit containing two heavy chains and optionally two light chains, so that the constant region of the heavy chain in the immunoglobulin unit is (2) producing an affinity substance-modified antibody or a salt thereof comprising a first modification moiety containing said first affinity substance therein; An affinity substance-modified antibody containing an affinity substance or a salt thereof is reacted with a compound of the present invention or a salt thereof to form the first and second modification moieties in the constant region of the heavy chain in the immunoglobulin unit.
  • the compound of the present invention or a salt thereof used in step (1) and the compound of the present invention or a salt thereof used in step (2) may be the same or different, and are preferably different.
  • the equivalent amount of the compound of the present invention or its salt relative to the antibody in the reaction varies depending on factors such as the compound of the present invention or its salt and the type of antibody, so particularly Although not limited, it is, for example, from 1 to 100, preferably from 2 to 80, more preferably from 4 to 60, even more preferably from 5 to 40, particularly preferably from 6 to 20.
  • Such a reaction can be appropriately carried out under conditions (mild conditions) that do not cause protein denaturation or decomposition (eg, cleavage of amide bonds).
  • mild conditions e.g, cleavage of amide bonds.
  • affinity-modified antibodies or salts thereof have the following formula (V): [During the ceremony, Ig refers to an immunoglobulin unit comprising two heavy chains consisting of a first and a second heavy chain and optionally two light chains; LL and LR each independently represent a linker, A L represents the first affinity substance, AR represents the second affinity substance, The average percentage modification r L of the immunoglobulin units by the first modifying moiety and the average percentage modification r R of the immunoglobulin units by the second modifying moiety are each from 65 to 135%. ] or a salt thereof may be used.
  • the affinity agent-modified antibody or salt thereof comprises (iii') a first cleavable moiety between (i') said first affinity agent and (ii') said immunoglobulin unit. and/or (iii'') a second cleavable moiety between (i'') the second affinity substance and (ii'') the immunoglobulin unit. good.
  • the first and second cuttable portions are similar to the cuttable portions described above. The first and second cuttable portions may be the same or different.
  • the cleavable moiety may be one that can generate a bioorthogonal functional group on the immunoglobulin unit side by cleavage.
  • cleavable moieties include, for example, disulfide residues, ester residues (including conventional ester residues, and other ester residues mentioned above such as thioester residues), acetal residues (conventional ester residues), and other acetal residues such as thioacetal residues), ketal residues, imine residues, and vicinal diol residues.
  • a compound represented by formula (Ia) or a salt thereof is combined with two heavy chains and optionally two light chains. This can be carried out by reacting with an antibody containing an immunoglobulin unit or a salt thereof.
  • the affinity substance-modified antibody or salt thereof has the following formula (Va-1): [During the ceremony, Ig indicates the immunoglobulin unit, W L1 , W L2 and W L3 , and W R1 , W R2 and W R3 each independently represent an oxygen atom or a sulfur atom, L L3 and L R3 each independently represent a third linker, L L4 and L R4 each independently represent a fourth linker, S represents a sulfur atom, A L represents the first affinity substance, AR represents the second affinity substance, The average percentage modification r L of the immunoglobulin units by the first modifying moiety and the average percentage modification r R of the immunoglobulin units by the second modifying moiety are each from 65 to 135%. ] or a salt thereof may be used.
  • a compound represented by formula (Ia-1) or a salt thereof is combined with two heavy chains and, if necessary, two heavy chains. This can be carried out by reacting with an antibody containing an immunoglobulin unit containing a light chain or a salt thereof.
  • the affinity substance-modified antibody or salt thereof also further comprises (iv') a first bioorthogonal functional group between (ii') the immunoglobulin unit and (iii') the first cleavable moiety; and/or (iv'') a second bioorthogonal functional group may be further included between (ii'') the immunoglobulin unit and (iii'') the second cleavable moiety.
  • the bioorthogonal functional group is as described above.
  • the bioorthogonal functional group is an azide residue, an alkyne residue (preferably a ring group having a triple bond between carbon atoms, which may be substituted with a substituent as described above), or a tetrazine residue. , alkene residues, thiol residues, maleimide residues, thiol residues, furan residues, and halocarbonyl residues.
  • the affinity substance-modified antibody or its salt further contains a bioorthogonal functional group between the immunoglobulin unit and the cleavable moiety
  • Vb the following formula (Vb):
  • Ig indicates the immunoglobulin unit
  • L L5 and L R5 each independently represent a fifth linker
  • L L6 and L R6 each independently represent a sixth linker
  • BL represents a first group containing a first bioorthogonal functional group
  • BR represents a second group containing a second bioorthogonal functional group
  • CLE L and CLE R each independently represent a cleavable moiety
  • a L represents the first affinity substance
  • AR represents the second affinity substance
  • the average percentage modification r L of the immunoglobulin units by the first modifying moiety and the average percentage modification r R of the immunoglobulin units by the second modifying moiety are each from 65 to 135%.
  • a salt thereof may be used.
  • a compound represented by formula (Ib) or a salt thereof is combined with two heavy chains and optionally two light chains. This can be carried out by reacting with an antibody containing an immunoglobulin unit or a salt thereof.
  • the affinity substance-modified antibody or salt thereof has the following formula (Vb-1): [During the ceremony, Ig indicates the immunoglobulin unit, W L1 , W L2 and W L3 , and W R1 , W R2 and W R3 each independently represent an oxygen atom or a sulfur atom, L L7 and L R7 each independently represent a seventh linker, L L8 and L R8 each independently represent an eighth linker, BL represents a first group containing a first bioorthogonal functional group, BR represents a second group containing a second bioorthogonal functional group, V L and V R each independently represent an oxygen atom or a sulfur atom, A L represents the first affinity substance, AR represents the second affinity substance, The average percentage modification r L of the immunoglobulin units by the first modifying moiety and the average percentage modification r R of the immunoglobulin units by the second modifying moiety are each from 65 to 135%. ] or a salt thereof may be used.
  • a compound represented by formula (Ib-1) or a salt thereof is combined with two heavy chains and optionally two This can be carried out by reacting with an antibody containing an immunoglobulin unit containing a light chain or a salt thereof.
  • the affinity substance-modified antibody or salt thereof also further comprises (iii') a first cleavable moiety between (i') the first affinity substance and (ii') the immunoglobulin unit, and ( It may further include (iv'') a first bioorthogonal functional group between the immunoglobulin unit (ii'') and (iii'') the second cleavable moiety.
  • the first cleavable moiety may be a cleavable moiety that can generate a second bioorthogonal functional group on the immunoglobulin unit side by cleavage.
  • the affinity substance-modified antibody or salt thereof has the following formula (Vc): [During the ceremony, Ig indicates the immunoglobulin unit, L R1 indicates the first linker, L R2 represents a second linker; L L5 indicates the fifth linker, L L6 indicates the sixth linker, BL represents a group containing the first bioorthogonal functional group, CLE L indicates the first cleavable portion; CLE(B) R represents a second cleavable moiety capable of generating a second bioorthogonal functional group on the immunoglobulin unit side by cleavage; A L represents the first affinity substance, AR represents the second affinity substance, The average percentage modification r L of the immunoglobulin units by the first modifying moiety and the average percentage modification r R of the immunoglobulin units by the second modifying moiety are each from 65 to 135%. ] or a salt thereof may be used.
  • an antibody or a salt thereof containing a structural unit represented by formula (Vc) a compound represented by formula (Ia) or a salt thereof, and a compound represented by formula (Ib) or a salt thereof are prepared by combining two This can be carried out by reacting with an antibody or a salt thereof containing an immunoglobulin unit containing a heavy chain and optionally two light chains.
  • the affinity substance-modified antibody or salt thereof has the following formula (Vc-1): [During the ceremony, Ig indicates the immunoglobulin unit, W L1 , W L2 and W L3 , and W R1 , W R2 and W R3 each independently represent an oxygen atom or a sulfur atom, L R3 each independently represents a third linker, L R4 each independently represents a fourth linker, L L7 each independently represents a seventh linker, L L8 each independently represents an eighth linker, BL represents a group containing the first bioorthogonal functional group, V L represents an oxygen atom or a sulfur atom, A L represents the first affinity substance, AR represents the second affinity substance, The average percentage modification r L of the immunoglobulin units by the first modifying moiety and the average percentage modification r R of the immunoglobulin units by the second modifying moiety are each from 65 to 135%. ] or a salt thereof may be used.
  • an antibody or a salt thereof containing a structural unit represented by formula (Vc-1) is carried out using a compound represented by formula (Ia-1) or a salt thereof, and a compound represented by formula (Ib-1) or This can be done by reacting the salt with an antibody or a salt thereof comprising an immunoglobulin unit comprising two heavy chains and optionally two light chains.
  • the subscript "L” for any symbol is conveniently used for symbols located on the left with respect to the immunoglobulin unit (Ig).
  • the subscript "R” for any symbol is conveniently used for symbols placed to the right of the immunoglobulin unit (Ig).
  • the meanings of the symbols with the subscripts "L” and “R” are the same as the meanings of the symbols without the subscripts "L” and "R.”
  • W L1 and W R1 are the same as W 1 and are preferably oxygen atoms.
  • W L2 and W R2 are the same as W 2 and are preferably oxygen atoms.
  • W L3 and W R3 are the same as W 3 and are preferably oxygen atoms.
  • the linkers denoted L L and L R are similar to the linkers denoted L.
  • the linkers represented by L L and L R may be the same or different, and are preferably different.
  • the first linker denoted L L1 and L R1 is similar to the first linker denoted L 1 .
  • the first linkers represented by L L1 and L R1 may be the same or different, and are preferably different.
  • the second linker denoted L L2 and L R2 is similar to the second linker denoted L 2 .
  • the second linkers represented by L L2 and L R2 may be the same or different, and are preferably different.
  • the third linker denoted L L3 and L R3 is similar to the third linker denoted L 3 .
  • the third linkers represented by L3 and L R3 may be the same or different, and are preferably different.
  • the fourth linker denoted L L4 and L R4 is similar to the fourth linker denoted L 4 .
  • the fourth linkers represented by L L4 and L R4 may be the same or different, and are preferably different.
  • the fifth linker denoted L L5 and L R5 is similar to the fifth linker denoted L 5 .
  • the first linkers represented by L L5 and L R5 may be the same or different, and are preferably different.
  • the sixth linker denoted L L6 and L R6 is similar to the sixth linker denoted L 6 .
  • the sixth linkers represented by L L6 and L R6 may be the same or different, and are preferably different.
  • the seventh linker designated L L7 and L R7 is similar to the seventh linker designated L 7 .
  • the seventh linkers represented by L L7 and L R7 may be the same or different, and are preferably different.
  • the eighth linker indicated by L L8 and L R8 is similar to the eighth linker indicated by L 8 .
  • the eighth linkers represented by L L8 and L R8 may be the same or different, and are preferably different.
  • the cleavable moieties denoted by CLE(B) L and CLE(B) R are similar to the cleavable moieties denoted by CLE(B).
  • the cleavable moieties represented by CLE(B) L and CLE(B) R may be the same or different, and are preferably different.
  • the first group containing the first bioorthogonal functional group represented by B L and the second group containing the second bioorthogonal functional group represented by B R are the bioorthogonal functional group represented by B.
  • the first group containing the first bioorthogonal functional group represented by B L and the second group containing the second bioorthogonal functional group represented by B R may be the same or different. Preferably, they are different.
  • the cleavable moieties designated CLE L and CLE R are similar to the cleavable moieties designated CLE.
  • the cleavable moieties represented by CLE L and CLE R may be the same or different, and are preferably different.
  • the affinity substances indicated by A L and A R are the same as the affinity substances indicated by A.
  • the affinity substances represented by A L and A R may be the same or different.
  • the degree of average modification percentage denoted by r L and r R and the method for determining it are the same as the average modification percentage denoted by r.
  • the average modification percentages represented by r L and r R may be the same or different, and are preferably different.
  • Confirmation of the production of the target affinity substance-modified antibody or its salt depends on the specific raw materials and the molecular weight of the product, but for example, electrophoresis, chromatography (e.g., gel filtration chromatography, ion exchange chromatography), etc. This can be carried out by chromatography, reverse phase column chromatography, HPLC), or mass spectrometry. Confirmation of position selectivity can be performed by peptide mapping. Peptide mapping can be performed, for example, by protease treatment and mass spectrometry. As the protease, endoprotease is preferred.
  • affinity substance-modified antibody or its salt can be appropriately purified by any method such as chromatography (eg, the above-mentioned chromatography and affinity chromatography).
  • the affinity substance-modified antibody or its salt may further contain an additional modification moiety.
  • an affinity substance-modified antibody or a salt thereof can be modified to further include an additional modification moiety. Additional modifying moieties may be introduced into the heavy chain or light chain of the antibody, preferably in the heavy chain of the antibody (particularly in the constant region of the heavy chain).
  • the additional modifying moiety may be an additional affinity agent that includes a fifth affinity moiety that has affinity for the constant region in the heavy chain of an antibody.
  • the "affinity moiety" in the "fifth affinity moiety” and the “affinity substance” in the “additional affinity substance” are each the same as described above.
  • the fifth affinity moiety may be the same or different from the first, second, third and/or fourth affinity moieties described above, but is preferably different.
  • an additional modifying moiety comprising a fifth affinity moiety having affinity for a constant region in a heavy chain of an antibody is present at one or more positions in the constant region of said two heavy chains. It may be introduced into the constant regions of the two heavy chains through modification of the amino groups in the side chains of lysine residues.
  • An affinity substance-modified antibody or a salt thereof is produced by combining two heavy chain constant regions (preferably Fc Additional modification moieties can be included through modification of the amino group in the side chain of one or more (preferably one or two, more preferably one) lysine residues in the CH2 domain or CH2 domain).
  • the position of one or more (preferably 1 or 2, more preferably 1) lysine residues is at position 246/248, 288/290, or 317 of the human IgG heavy chain according to EU numbering.
  • the position at which the additional modification moiety, including the fifth affinity moiety having affinity for the constant region in the heavy chain of the antibody, is introduced is located at the first and second affinity moieties having affinity for the constant region in the heavy chain of the antibody.
  • a first modifying moiety comprising a first affinity agent comprising a sex moiety, and a second affinity agent comprising third and fourth affinity portions having affinity for a constant region in a heavy chain of an antibody.
  • the position is different from the position at which the second modification moiety is introduced.
  • the positions where both the first modification moiety and the second modification moiety are introduced are lysine residues at positions 246/248
  • the positions where the additional modification moiety is introduced are positions 288/290.
  • lysine residues at position 317 are preferred, and lysine residues at positions 288/290 are more preferred.
  • positions at which both the first modification moiety and the second modification moiety are introduced are lysine residues at positions 288/290
  • the positions at which the above additional modification moieties are introduced are at positions 246/248, or Lysine residues at position 317 are preferred, and lysine residues at positions 246/248 are more preferred. If the position at which both the first modification moiety and the second modification moiety are introduced is the lysine residue at position 317, the position at which the additional modification moiety is introduced is at position 246/248, or position 288/ A lysine residue at position 290 is preferred.
  • Antibody or salt thereof without affinity substance 5-1 An antibody or a salt thereof that does not contain an affinity substance contains (A) an affinity substance that includes first and second affinity portions that have an affinity for the constant region in the heavy chain of the antibody (immunoglobulin unit), and ( B) An affinity substance-modified antibody or a salt thereof that contains an antibody (immunoglobulin unit) and (C) further contains a cleavable moiety between (A) the affinity substance and (B) the antibody (immunoglobulin unit). It can be manufactured using
  • the method for producing an antibody or a salt thereof that does not contain an affinity substance may be the following method 1-1 or 1-2.
  • Method 1-1 (A) an affinity substance comprising first and second affinity portions that have affinity for the constant region in the heavy chain of an antibody (immunoglobulin unit); ) and (C) further comprising a cleavable moiety between (A) the affinity substance and (B) the antibody (immunoglobulin unit), or a salt thereof, which is cleaved with the cleavable moiety.
  • a method for producing an antibody or a salt thereof free of an affinity substance the method comprising processing to produce an antibody or a salt thereof free of an affinity substance.
  • a method for producing an antibody or a salt thereof that does not contain an affinity substance including the following (1) and (2): (1) (A) An affinity substance comprising first and second affinity parts having affinity for the constant region in the heavy chain of an antibody (immunoglobulin unit), and (B) reaction against the antibody (immunoglobulin unit) and (C) a compound or a salt thereof that further contains a cleavable moiety between (A) the affinity substance and (B) the reactive group, between two heavy chains and, if necessary, reacting with an antibody or a salt thereof comprising an immunoglobulin unit comprising two light chains to produce an affinity substance-modified antibody or a salt thereof comprising a cleavable moiety between the affinity substance and the antibody; and (2) an affinity substance-modified antibody or a salt thereof containing a cleavable moiety between the affinity substance and the antibody is cleaved with the cleavage moiety to produce an antibody or a salt thereof that does not contain the affinity substance
  • the cutting treatment includes (a) treatment with one or more substances selected from the group consisting of acidic substances, basic substances, reducing agents, oxidizing agents, and enzymes as described above, (b) physicochemical stimulation such as light. or (c) incubation when using a cleavable linker containing a self-degradable cleavable moiety.
  • treatment with one or more substances selected from the group consisting of acidic substances, basic substances, reducing agents, oxidizing agents, and enzymes as described above, (b) physicochemical stimulation such as light. or (c) incubation when using a cleavable linker containing a self-degradable cleavable moiety.
  • Such a cleavage reaction can be appropriately carried out under conditions (mild conditions) that do not cause protein denaturation or decomposition (eg, cleavage of amide bonds).
  • mild conditions are as described above.
  • the cleavage reaction can be performed using a hydroxylamine hydrochloride solution (e.g., pH 4.0-8.0, 10mM-10M). This can be done by incubating for an appropriate time (e.g., 1 hour) in a microorganism (e.g., Vance, N. et al., Bioconjugate Chem. 2019, 30, 148-160).
  • Confirmation of the production of an antibody or its salt that does not contain an affinity substance obtained by the cleavage reaction depends on the specific raw materials and the molecular weight of the product, but can be confirmed by, for example, electrophoresis, chromatography (e.g., gel filtration). This can be carried out by chromatography, ion exchange chromatography, reversed phase column chromatography (HPLC), or mass spectrometry. Regioselectivity can be confirmed by peptide mapping as described above. The number of introduced affinity substances can be confirmed by mass spectrometry (DAR calculator (Agilent software) can be used in combination).
  • the affinity substance-modified antibody or its salt can be appropriately purified by any method such as chromatography (eg, the above-mentioned chromatography and affinity chromatography).
  • an affinity substance containing first and second affinity parts that have affinity for the constant region in the heavy chain of an antibody (immunoglobulin unit) and (B) an antibody (immunoglobulin unit) When the affinity substance-modified antibody or its salt containing a cleavable moiety contains (a) a cleavable moiety that can generate a bioorthogonal functional group on the antibody (immunoglobulin unit) side by cleavage, or (b) When a bioorthogonal functional group is contained between the antibody (immunoglobulin unit) and the cleavable moiety, an antibody derivative containing a bioorthogonal functional group or its salt is used as an antibody or its salt that does not contain an affinity substance. Salt can be manufactured.
  • an antibody derivative containing a bioorthogonal functional group or a salt thereof with a functional substance, a conjugate of an antibody and a functional substance or a salt thereof is produced as an antibody or a salt thereof that does not contain an affinity substance. be able to.
  • An antibody derivative or a salt thereof containing at least one bioorthogonal functional group The present invention relates to (a) a constitutional unit of an antibody (an immunoglobulin unit containing two heavy chains and optionally two light chains); and (b) contains a bioorthogonal functional group, and (c) the bioorthogonal functional group is introduced into only the constant region of one of the heavy chains in the immunoglobulin unit (i.e., one of the heavy chains in the immunoglobulin unit). (a bioorthogonal functional group is introduced into the constant region of one heavy chain and no bioorthogonal functional group is introduced into the constant region of the other heavy chain), an antibody derivative containing a bioorthogonal functional group or its Provide salt. Definitions, examples, and preferred examples of antibodies, immunoglobulin units, and bioorthogonal functional groups, and their constituent elements (eg, constant regions) are as described above.
  • the antibody derivative or its salt contains asparagine, glutamine, methionine, proline, serine, threonine, tryptophan, tyrosine, aspartic acid, glutamic acid, arginine, histidine, and lysine present in the constant region (preferably Fc region or CH2 domain). Containing a bioorthogonal functional group through modification of the functional group in the side chain of any one or two or more (e.g., 2, 3, 4) of the 14 amino acid residues consisting of I can do it.
  • the antibody derivative or a salt thereof is preferably modified with a functional group in the side chain of any one of lysine, tyrosine, tryptophan, or cysteine present in the constant region (preferably Fc region or CH2 domain). More preferably via modification of a functional group in the side chain of any one of the amino acids lysine, tyrosine, or tryptophan, even more preferably via modification of a functional group in the side chain of lysine or tyrosine.
  • Bioorthogonal functional groups can be included via modification, particularly preferably via modification of the amino group in the side chain of lysine. The positions of these amino acid residues in the constant region are as described above.
  • the modification position of an antibody or its salt with a bioorthogonal functional group can be confirmed by peptide mapping. Modifications may be regioselective, as described above. Therefore, in formulas (IIIa), (IIIa-1), (IIIb), and (IIIb-1) described below, the immunoglobulin unit is modified by the corresponding modification via the functional group in the side chain of the above amino acid residue. It may have units regioselectively.
  • the antibody derivative comprises a constant region (preferably an Fc region or a CH2 domain) of one heavy chain in an antibody building block (an immunoglobulin unit comprising two heavy chains and optionally two light chains).
  • an antibody building block an immunoglobulin unit comprising two heavy chains and optionally two light chains.
  • bioorthogonal functional groups in other words, through modification of the amino group in the side chain of one or more (preferably one or two, more preferably one) lysine residues in containing a bioorthogonal functional group through the amino group in the side chain of a lysine residue in the constant region of one heavy chain in the globulin unit; does not contain bioorthogonal functional groups via amino groups).
  • the position of one or more (preferably 1 or 2, more preferably 1) lysine residues is at position 246/248, 288/290, or 317 of the human IgG heavy chain according to EU numbering.
  • Modifications may be regioselective, as described above. Therefore, in formulas (IIIa), (IIIa-1), (IIIb), and (IIIb-1) described below, the immunoglobulin unit is modified by the corresponding modification via the amino group in the side chain of the lysine residue. It may have units regioselectively.
  • the antibody derivative or salt thereof has the following formula (IIIa): [During the ceremony, Ig refers to an immunoglobulin unit containing two heavy chains and optionally two light chains; L 1 indicates the first linker, B represents a group containing a bioorthogonal functional group, The average percentage modification r of the immunoglobulin units by bioorthogonal functional groups is between 65 and 135%. ] or a salt thereof may be used.
  • the immunoglobulin unit denoted Ig, the first linker denoted L1 , the group containing a bioorthogonal functional group denoted B, and the average modification percentage denoted r, as well as definitions, examples, and preferred examples of antibodies are: , as described above.
  • a particularly preferred bioorthogonal functional group is a thiol group.
  • the molecular weight of the partial structure represented by L 1 -B may be 700 or less.
  • the antibody derivative having a bioorthogonal functional group or a salt thereof has a very small ratio of the molecular weight of the partial structure to the molecular weight of the entire antibody.
  • purification based on differences in molecular weight is relatively difficult, according to the present invention, which enables advanced control of DAR, antibody derivatives exhibiting a desired DAR can be obtained without necessarily requiring purification based on differences in molecular weight. Can be obtained in high purity.
  • the molecular weight of the partial structure represented by L 1 -B is preferably 600 or less, more preferably 500 or less, even more preferably 400 or less, particularly preferably 300 or less, 250 or less, 200 or less, or It may be 100 or less.
  • the antibody derivative or salt thereof has the following formula (IIIa-1): [During the ceremony, Ig refers to an immunoglobulin unit containing two heavy chains and optionally two light chains; W 1 represents an oxygen atom or a sulfur atom, L 3 indicates a third linker, SH represents a thiol group, The average percentage modification r of the immunoglobulin units by bioorthogonal functional groups is between 65 and 135%. ] or a salt thereof may be used.
  • the immunoglobulin unit designated Ig, the atom designated W1 , the third linker designated L3 , and the average modification percentage designated r, as well as the definition, examples, and preferred examples of antibodies are as described above. .
  • the third linker represented by L 3 may be (CH 2 ) n1 .
  • n1 is an integer from 1 to 10.
  • n1 may be an integer of 2 or more.
  • n1 may also be an integer of 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less.
  • n1 is 2.
  • the antibody derivative or salt thereof has the following formula (IIIb): [During the ceremony, Ig refers to an immunoglobulin unit containing two heavy chains and optionally two light chains; L 5 indicates the fifth linker, B represents a group containing a bioorthogonal functional group, T 1 represents a monovalent group, The average percentage modification r of the immunoglobulin units by bioorthogonal functional groups is between 65 and 135%. ] or a salt thereof may be used.
  • the immunoglobulin unit denoted Ig, the fifth linker denoted L5 , the group containing a bioorthogonal functional group denoted B, and the average modification percentage denoted r, as well as definitions, examples, and preferred examples of antibodies are: , as described above.
  • a particularly preferred bioorthogonal functional group is an azide group.
  • T 1 is a monovalent group and can be generated by cleaving a cleavable moiety.
  • a monovalent group may be substituted or unsubstituted. Examples of the monovalent group include those mentioned above. Examples of the substituent when a monovalent group is substituted include those mentioned above.
  • the monovalent group represented by T 1 may be an optionally substituted hydroxyamino group.
  • the optionally substituted hydroxyamino group can be represented by the following formula ( ⁇ ). NR i - OR ii ( ⁇ ) [During the ceremony, R i and R ii each independently represent a hydrogen atom or a monovalent hydrocarbon group. )
  • the monovalent hydrocarbon group may be substituted or unsubstituted.
  • the definition, examples, and preferred examples of the monovalent hydrocarbon group and the substituent when the monovalent hydrocarbon group is substituted are as described above.
  • the optionally substituted hydroxyamino group may be NH-OR ii (where R ii represents an alkyl group). More preferably, the optionally substituted hydroxyamino group may be NH-OR ii (wherein R ii represents an alkyl group having 1 to 6 carbon atoms).
  • the molecular weight of the partial structure represented by L 5 (-B)-T 1 may be 700 or less.
  • the molecular weight of the partial structure represented by L 5 (-B)-T 1 is preferably 600 or less, more preferably 500 or less, even more preferably 400 or less, particularly preferably 300 or less, and 250 or less. , 200 or less, or 100 or less.
  • the antibody derivative or salt thereof has the following formula (IIIb-1): [During the ceremony, Ig refers to an immunoglobulin unit containing two heavy chains and optionally two light chains; W 1 and W 2 each independently represent an oxygen atom or a sulfur atom, L 7 indicates the seventh linker, B represents a group containing a bioorthogonal functional group, T 2 represents a monovalent group, The modification percentage r of the immunoglobulin units with bioorthogonal functional groups is between 65 and 135%. ] or a salt thereof may be used.
  • an immunoglobulin unit denoted Ig
  • atoms denoted W 1 and W 2
  • a seventh linker denoted L 7
  • a group containing a bioorthogonal functional group denoted B
  • an average modification percentage denoted r;
  • a particularly preferred bioorthogonal functional group is an azide group.
  • T 2 is a monovalent group and can be generated by cleaving a cleavable moiety.
  • a monovalent group may be substituted or unsubstituted. Examples of the monovalent group include those mentioned above. Examples of the substituent when a monovalent group is substituted include those mentioned above.
  • the monovalent group represented by T 2 may be an optionally substituted hydroxyamino group. Details of the optionally substituted hydroxyamino group are the same as those described for T1 .
  • the seventh linker represented by L 7 may be (CH 2 ) n2 .
  • n2 is an integer from 1 to 10.
  • n2 may be an integer of 2 or more, or 3 or more.
  • n2 may also be an integer of 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, or 3 or less.
  • n2 may be 3.
  • n3 is an integer from 1 to 10.
  • n3 may be an integer of 2 or more, 3 or more, or 4 or more.
  • n3 may also be an integer of 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, or 4 or less.
  • n3 may be 4.
  • an antibody derivative or a salt thereof containing a bioorthogonal functional group involves (A) an affinity substance containing a first and second affinity moiety having affinity for the constant region in the heavy chain of an antibody (immunoglobulin unit); , and (B) an antibody (immunoglobulin unit), and (C) an affinity substance-modified antibody further comprising a cleavable moiety between (A) the affinity substance and (B) the antibody (immunoglobulin unit). Or it can be carried out using its salt.
  • the affinity substance-modified antibody or a salt thereof includes (a) a cleavable moiety that can generate a bioorthogonal functional group on the antibody (immunoglobulin unit) side by cleavage, the bioorthogonal functional Antibody derivatives or salts thereof containing the group can be produced.
  • such manufacturing methods include, for example, the following (2-1) to (2-6) (FIGS. 2 to 6).
  • Method 2-1 (A) an affinity substance comprising first and second affinity parts having affinity for the constant region in the heavy chain of an antibody (immunoglobulin unit); ), and (C) a cleavable moiety between (A) the affinity substance and (B) the antibody (immunoglobulin unit) (herein, the cleavable moiety is a cleavable moiety that binds the bioorthogonal functional group to the antibody by cleavage); (a cleavable moiety that can be generated on the immunoglobulin unit side) or a salt thereof is cleaved with the cleavable moiety to produce an antibody derivative or a salt thereof containing a bioorthogonal functional group.
  • a method for producing an antibody derivative or a salt thereof containing a bioorthogonal functional group the method comprising producing a bioorthogonal functional group-containing antibody derivative or a salt thereof.
  • Method 2-2 A method for producing an antibody derivative or a salt thereof containing a bioorthogonal functional group, including the following (1) and (2): (1) (A) an affinity substance comprising first and second affinity parts having affinity for the constant region in the heavy chain of an antibody (immunoglobulin unit), and (B) reaction to the antibody (immunoglobulin unit) (C) a cleavable moiety between (A) the affinity substance and (B) the reactive group; A compound or a salt thereof further comprising a cleavable moiety (which is a cleavable moiety that can be generated on the side of a reactive group for an immunoglobulin unit) containing an immunoglobulin unit containing two heavy chains and optionally two light chains.
  • a cleavable moiety is formed between the affinity substance and the antibody by reacting with the antibody or a salt thereof (herein, the cleavable moiety is a cleavable moiety that can generate a bioorthogonal functional group on the antibody side by cleavage). and (2) producing an affinity substance-modified antibody or a salt thereof comprising a cleavable moiety between the affinity substance and the antibody; cleavage at a moiety to produce an antibody derivative or a salt thereof containing a bioorthogonal functional group.
  • An antibody derivative or a salt thereof containing the structural unit represented by the above formula (IIIa) is obtained by cleaving an antibody containing the structural unit represented by the above formula (IIa) or a salt thereof with a cleavable moiety.
  • Method 2-4 A method for producing an antibody derivative or a salt thereof containing a bioorthogonal functional group, including the following (1) and (2): (1) A compound represented by the above formula (Ia) or a salt thereof is reacted with an antibody or a salt thereof containing an immunoglobulin unit containing two heavy chains and optionally two light chains, and the above-mentioned producing an antibody or a salt thereof containing a structural unit represented by formula (IIa); and (2) cleaving the antibody or a salt thereof containing a structural unit represented by formula (IIa) with a cleavable moiety; to produce an antibody derivative or a salt thereof containing a structural unit represented by the above formula (IIIa).
  • An antibody or a salt thereof containing the structural unit represented by the above formula (IIa-1) is cleaved with a cleavable moiety to obtain the structural unit represented by the above formula (IIIa-1).
  • a method for producing an antibody derivative or a salt thereof containing a bioorthogonal functional group the method comprising producing an antibody derivative or a salt thereof containing a bioorthogonal functional group.
  • Method 2-6 A method for producing an antibody derivative or a salt thereof containing a bioorthogonal functional group, including the following (1) and (2): (1) A compound represented by the above formula (Ia-1) or a salt thereof is reacted with an antibody containing an immunoglobulin unit containing two heavy chains and optionally two light chains or a salt thereof. , producing an antibody or a salt thereof comprising a structural unit represented by the above formula (IIa-1); and (2) producing an antibody or a salt thereof comprising a structural unit represented by the above formula (IIa-1), Cleavage treatment with a cleavable moiety to produce an antibody derivative or a salt thereof containing a structural unit represented by the above formula (IIIa-1).
  • the above method 2-1 may be performed by the above method 2-3 or 2-5.
  • the above method 2-2 may be performed by the above method 2-4 or 2-6.
  • the above methods 2-2, 2-4, and 2-6 involve producing a compound of the present invention or a salt thereof by reacting an affinity substance of the present invention with a partial compound containing a reactive group for an antibody. It may further contain (FIGS. 2 to 6).
  • the affinity substance-modified antibody or salt thereof includes (b) a bioorthogonal functional group between the antibody (immunoglobulin unit) and the cleavable moiety
  • the bioorthogonal functional group Antibody derivatives or salts thereof containing the group can be produced.
  • such manufacturing methods include, for example, the following (2-7) to (2-12) (FIGS. 2 to 6).
  • Method 2--7 (A) an affinity substance comprising first and second affinity portions having affinity for the constant region in the heavy chain of an antibody (immunoglobulin unit); ), and (C) a cleavable moiety between (A) the affinity substance and (B) the antibody (immunoglobulin unit), and (D) the cleavable moiety between the antibody (immunoglobulin unit) and A bioorthogonal method comprising cleaving an affinity substance-modified antibody or a salt thereof further comprising a bioorthogonal functional group with a cleavable moiety to produce an antibody derivative or a salt thereof comprising a bioorthogonal functional group.
  • a method for producing an antibody derivative or a salt thereof containing a functional group A method for producing an antibody derivative or a salt thereof containing a functional group.
  • Method 2-8 A method for producing an antibody derivative containing a bioorthogonal functional group or a salt thereof, including the following (1) and (2): (1) (A) an affinity substance comprising first and second affinity parts having affinity for the constant region in the heavy chain of an antibody (immunoglobulin unit), and (B) reaction to the antibody (immunoglobulin unit) and (C) a cleavable moiety between (A) the affinity substance and (B) the reactive group, and (D) a bioorthogonal moiety between the reactive group and the cleavable moiety.
  • a compound further containing a sexual functional group or a salt thereof is reacted with an antibody containing an immunoglobulin unit containing two heavy chains and optionally two light chains or a salt thereof, thereby producing the above-mentioned affinity substance and the above-mentioned antibody.
  • An affinity substance-modified antibody or a salt thereof containing a cleavage moiety between the affinity substance and the antibody is cleaved with the cleavage moiety to produce an antibody derivative or a salt thereof containing a bioorthogonal functional group.
  • An antibody derivative or a salt thereof containing the structural unit represented by the above formula (IIIb) is obtained by cleaving an antibody containing the structural unit represented by the above formula (IIb) or a salt thereof with a cleavable moiety.
  • Method 2-10) A method for producing an antibody derivative or a salt thereof containing a bioorthogonal functional group, including the following (1) and (2): (1) A compound represented by the above formula (Ib) or a salt thereof is reacted with an antibody or a salt thereof containing an immunoglobulin unit containing two heavy chains and optionally two light chains, and the above-mentioned producing an antibody or a salt thereof containing a structural unit represented by formula (IIb); and (2) cleaving the antibody or a salt thereof containing a structural unit represented by formula (IIb) with a cleavable moiety; to produce an antibody derivative or a salt thereof containing a structural unit represented by the above formula (IIIb).
  • An antibody or a salt thereof containing the structural unit represented by the above formula (IIb-1) is cleaved with a cleavable moiety to obtain the structural unit represented by the above formula (IIIb-1).
  • a method for producing an antibody derivative or a salt thereof containing a bioorthogonal functional group the method comprising producing an antibody derivative or a salt thereof containing a bioorthogonal functional group.
  • Method 2-12 A method for producing an antibody derivative or a salt thereof containing a bioorthogonal functional group, including the following (1) and (2): (1) A compound represented by the above formula (Ib-1) or a salt thereof is reacted with an antibody containing an immunoglobulin unit containing two heavy chains and optionally two light chains or a salt thereof. , producing an antibody or a salt thereof comprising a structural unit represented by the above formula (IIb-1); and (2) producing an antibody or a salt thereof comprising a structural unit represented by the above formula (IIb-1), Cleavage treatment with a cleavable moiety to produce an antibody derivative or a salt thereof containing a structural unit represented by the above formula (IIIb-1).
  • the above method 2-7 may be performed by the above method 2-9 or 2-11.
  • the above method 2-8 may be performed by the above method 2-10 or 2-11.
  • Methods 2-8, 2-10, and 2-12 above further include reacting the affinity substance of the invention with a moiety containing a reactive group for an antibody to produce a compound of the invention or a salt thereof. ( Figures 2 to 6).
  • the antibody derivative or its salt may further contain additional modification moieties.
  • additional modification moieties may be introduced into the heavy chain or light chain of the antibody, preferably in the heavy chain of the antibody (particularly in the constant region of the heavy chain).
  • the additional modifying moiety may be an additional modifying moiety that includes a bioorthogonal functional group.
  • the bioorthogonal functional groups are the same as those described above.
  • the bioorthogonal functional group contained in the additional modification moiety may be the same as or different from the bioorthogonal functional group in (b) above, but is preferably different.
  • the additional modifying moiety comprising a bioorthogonal functional group modifies an amino group in a side chain of a lysine residue present at one or more positions in the constant region of said two heavy chains.
  • the position of one or more (preferably 1 or 2, more preferably 1) lysine residues is at position 246/248, 288/290, or 317 of the human IgG heavy chain according to EU numbering.
  • the position where the additional modification moiety containing the bioorthogonal functional group is introduced is preferably different from the position where the bioorthogonal functional group described in (b) above is introduced.
  • the above additional modification moiety is introduced into the lysine residue at positions 288/290 or 317. Lysine residues at positions 288/290 are preferred, and lysine residues at positions 288/290 are more preferred.
  • the additional modification moiety is introduced at the lysine residue at position 246/248 or 317. residues are preferred, and lysine residues at positions 246/248 are more preferred.
  • the additional modification moiety is introduced to the lysine residue at positions 246/248 or 288/290. Residues are preferred.
  • An antibody derivative or a salt thereof comprising at least two bioorthogonal functional groups
  • the present invention also provides an antibody derivative comprising: (a) two heavy chains consisting of a first and a second heavy chain and optionally two light chains; an immunoglobulin unit, and (b) a first modification moiety comprising a first bioorthogonal functional group and a second modification moiety comprising a second bioorthogonal functional group; (c) a first modification moiety is introduced into the constant region of the first heavy chain; (d) a second modification moiety is introduced into the constant region of the second heavy chain, and (e) the first and second modification moieties are different from each other; An antibody derivative or a salt thereof is provided. Definitions, examples, and preferred examples of antibodies, immunoglobulin units, and bioorthogonal functional groups, and their constituent elements (eg, constant regions) are as described above.
  • the antibody derivative or its salt contains asparagine, glutamine, methionine, proline, serine, threonine, tryptophan, tyrosine, aspartic acid, glutamic acid, arginine, histidine, and lysine present in the constant region (preferably Fc region or CH2 domain).
  • the antibody derivative or a salt thereof is preferably modified with a functional group in the side chain of any one of lysine, tyrosine, tryptophan, or cysteine present in the constant region (preferably Fc region or CH2 domain).
  • a first modification moiety comprising a first bioorthogonal functional group, and a second bioorthogonal functional group, through modification, particularly preferably through modification of an amino group in the side chain of lysine.
  • a second modifying moiety can be included.
  • the positions of these amino acid residues in the constant region are as described above.
  • the modification position of an antibody or its salt with a bioorthogonal functional group can be confirmed by peptide mapping. Modifications may be regioselective, as described above. Therefore, in the below-mentioned formula, the immunoglobulin unit may have a corresponding modification unit regioselectively via the functional group in the side chain of the amino acid residue.
  • the antibody derivative comprises a constant region (preferably an Fc region or a CH2 domain) of one heavy chain in an antibody building block (an immunoglobulin unit comprising two heavy chains and optionally two light chains). ) respectively comprising a first bioorthogonal functional group via modification of an amino group in the side chain of one or more (preferably one or two, more preferably one) lysine residues in moiety, and a second modified moiety that includes a second bioorthogonal functional group. More specifically, the position of one or more (preferably 1 or 2, more preferably 1) lysine residues is at position 246/248, 288/290, or 317 of the human IgG heavy chain according to EU numbering. There may be.
  • Modifications may be regioselective, as described above. Therefore, in the below-mentioned formula, the immunoglobulin unit may have a corresponding modification unit regioselectively via the amino group in the side chain of the lysine residue.
  • the antibody derivative or salt thereof has the following formula (VIa): [During the ceremony, Ig indicates the immunoglobulin unit, L L1 and L R1 each independently represent a first linker, BL represents a first group containing a first bioorthogonal functional group, BR represents a second group containing a second bioorthogonal functional group, The average percentage modification r L of said immunoglobulin units by the first modifying moiety and the average percentage modification r R of said immunoglobulin units by the second modifying moiety are each between 65 and 135%. ] or a salt thereof may be used.
  • the production of an antibody or a salt thereof containing a structural unit represented by formula (VIa) involves the preparation of CLE(B) L and CLE(B) in an affinity substance-modified antibody or a salt thereof containing a structural unit represented by formula (Va). ) This can be done by cutting two cleavable moieties represented by R. When the two cleavable moieties are the same, the cleavage reaction can be performed in one cleavage reaction.
  • the cleavage reaction can be a single cleavage reaction (e.g., when the two different cleavable moieties are cleavable with the same cleavage treatment or agent), or two cleavage reactions ( For example, if two different cuttable parts are cuttable with different cutting processes or cutting agents). Details of the cleavage reaction are as described above.
  • the antibody derivative or salt thereof has the following formula (VIa-1): [During the ceremony, Ig indicates the immunoglobulin unit, W L1 and W R1 each independently represent an oxygen atom or a sulfur atom, L L3 and L R3 each independently represent a third linker, SH represents a thiol group, which is a bioorthogonal functional group, The average percentage modification r L of said immunoglobulin units by the first modifying moiety and the average percentage modification r R of said immunoglobulin units by the second modifying moiety are each between 65 and 135%. ] or a salt thereof may be used.
  • an antibody containing a structural unit represented by formula (VIa-1) or a salt thereof can be carried out by using an affinity substance-modified antibody containing a structural unit represented by formula (Va-1) or a salt thereof represented by This can be done by cutting two cuttable parts.
  • the cleavage reaction can be carried out in one cleavage reaction. Details of the cleavage reaction are as described above.
  • the antibody derivative or salt thereof has the following formula (VIb): [During the ceremony, Ig indicates the immunoglobulin unit, L L5 and L R5 each independently represent a fifth linker, BL represents a first group containing a first bioorthogonal functional group, BR represents a second group containing a second bioorthogonal functional group, T L1 and T R1 each independently represent a monovalent group, The average percentage modification r L of said immunoglobulin units by the first modifying moiety and the average percentage modification r R of said immunoglobulin units by the second modifying moiety are each between 65 and 135%. ] or a salt thereof may be used.
  • an antibody containing a structural unit represented by formula (VIb) or a salt thereof can be carried out using 2, represented by CLE L and CLE R , in an affinity substance-modified antibody or a salt thereof containing a structural unit represented by formula (Vb). This can be done by cutting two cuttable parts. When the two cleavable moieties are the same, the cleavage reaction can be performed in one cleavage reaction.
  • the cleavage reaction can be a single cleavage reaction (e.g., when the two different cleavable moieties are cleavable with the same cleavage treatment or agent), or two cleavage reactions ( For example, if two different cuttable parts are cuttable with different cutting processes or cutting agents). Details of the cleavage reaction are as described above.
  • the antibody derivative or salt thereof has the following formula (VIb-1): [During the ceremony, Ig indicates the immunoglobulin unit, W L1 and W L2 and W R1 and W R2 each independently represent an oxygen atom or a sulfur atom, L L7 and L R7 each independently represent a seventh linker, BL represents a first group containing a first bioorthogonal functional group, BR represents a second group containing a second bioorthogonal functional group, T L2 and T R2 each independently represent a monovalent group, The average percentage modification r L of said immunoglobulin units by the first modifying moiety and the average percentage modification r R of said immunoglobulin units by the second modifying moiety are each between 65 and 135%. ] or a salt thereof may be used.
  • the production of an antibody or a salt thereof containing a structural unit represented by formula (VIb-1) involves C-V L and This can be done by cleaving two cleavable moieties represented by C-V R.
  • the cleavage reaction can be performed in one cleavage reaction.
  • the cleavage reaction can be a single cleavage reaction (e.g., when the two different cleavable moieties are cleavable with the same cleavage treatment or agent), or two cleavage reactions ( For example, if two different cuttable parts are cuttable with different cutting processes or cutting agents). Details of the cleavage reaction are as described above.
  • the antibody derivative or salt thereof has the following formula (VIc): [During the ceremony, Ig indicates the immunoglobulin unit, L R1 indicates the first linker, L L5 indicates the fifth linker, BL represents a first group containing a first bioorthogonal functional group, BR represents a second group containing a second bioorthogonal functional group, T L1 represents a monovalent group, The average percentage modification r L of said immunoglobulin units by the first modifying moiety and the average percentage modification r R of said immunoglobulin units by the second modifying moiety are each between 65 and 135%. ] or a salt thereof may be used.
  • an antibody containing a structural unit represented by formula (VIc) or a salt thereof is performed using CLE L and CLE(B) R in an affinity substance-modified antibody or a salt thereof containing a structural unit represented by formula (Vc). This can be done by cutting the two cuttable parts shown. When the two cleavable moieties are the same, the cleavage reaction can be performed in one cleavage reaction.
  • the cleavage reaction can be a single cleavage reaction (e.g., when the two different cleavable moieties are cleavable with the same cleavage treatment or agent), or two cleavage reactions ( For example, if two different cuttable parts are cuttable with different cutting processes or cutting agents). Details of the cleavage reaction are as described above.
  • the antibody derivative or salt thereof has the following formula (VIc-1): [During the ceremony, Ig indicates the immunoglobulin unit, W L1 and W L2 and W R1 each independently represent an oxygen atom or a sulfur atom, L R3 represents a third linker, L L7 indicates the seventh linker, BL represents a first group containing a first bioorthogonal functional group, SH represents a thiol group, which is a second bioorthogonal functional group, T L2 represents a monovalent group, The average percentage modification r L of said immunoglobulin units by the first modifying moiety and the average percentage modification r R of said immunoglobulin units by the second modifying moiety are each between 65 and 135%. ] or a salt thereof may be used.
  • the production of an antibody or a salt thereof containing a structural unit represented by formula (VIc-1) involves C-V L and This can be done by cutting two cleavable portions denoted by CS.
  • the cleavage reaction can be performed in one cleavage reaction.
  • the cleavage reaction can be a single cleavage reaction (e.g., when the two different cleavable moieties are cleavable with the same cleavage treatment or agent), or two cleavage reactions ( For example, if two different cuttable parts are cuttable with different cutting processes or cutting agents). Details of the cleavage reaction are as described above.
  • the third linkers represented by L L3 and L R3 may be the same or different, and are preferably different.
  • the fifth linker denoted L L5 and L R5 is similar to the fifth linker denoted L 5 .
  • the first linkers represented by L L5 and L R5 may be the same or different, and are preferably different.
  • the seventh linker designated L L7 and L R7 is similar to the seventh linker designated L 7 .
  • the seventh linkers represented by L L7 and L R7 may be the same or different, and are preferably different.
  • the first group containing the first bioorthogonal functional group represented by B L and the second group containing the second bioorthogonal functional group represented by B R are the bioorthogonal functional group represented by B.
  • the first group containing the first bioorthogonal functional group represented by B L and the second group containing the second bioorthogonal functional group represented by B R may be the same or different. Preferably, they are different.
  • the monovalent groups represented by T L1 and T R1 are the same as the monovalent group represented by T 1 .
  • the monovalent groups represented by T L1 and T R1 may be the same or different.
  • the monovalent groups represented by T L2 and T R2 are the same as the monovalent group represented by T 2 .
  • the monovalent groups represented by T L2 and T R2 may be the same or different.
  • the degree of average modification percentage denoted by r L and r R and the method for determining it are the same as the average modification percentage denoted by r.
  • the average modification percentages represented by r L and r R may be the same or different, and are preferably different.
  • the molecular weight of the substructure represented by L L1 -B L and/or L R1 -B R in formulas (VIa) and (VIc) is the same as that represented by L 1 -B in formula (IIIa).
  • the molecular weight may be the same as the above molecular weight of the partial structure.
  • the third linker represented by L L3 and/or L R3 in formulas (VIa-1) and (VIc-1) is the third linker represented by L 3 in formula (IIIa-1). It may be the same as the linker (CH 2 ) n1 .
  • the monovalent group represented by T L1 , T R1 , T L2 , and/or T R2 is , T 1 and/or T 2 in formula (IIIb), which may be an optionally substituted hydroxyamino group.
  • the molecular weight of the substructure represented by L L5 (-B L )-T L1 and/or L R5 (-B R )-T R1 is The molecular weight may be the same as that of the partial structure represented by L 5 (-B)-T 1 in (IIIb).
  • the seventh linker represented by L L7 and/or L R7 in formulas (VIb-1) and (VIc-1) is the seventh linker represented by L 7 in formula (IIIb-1). It may be the same as the linker (CH 2 ) n2 .
  • the antibody derivative or its salt may further contain additional modification moieties.
  • additional modification moieties may be introduced into the heavy chain or light chain of the antibody, preferably in the heavy chain of the antibody (particularly in the constant region of the heavy chain).
  • the additional modifying moiety may be an additional modifying moiety that includes a bioorthogonal functional group.
  • the bioorthogonal functional groups are the same as those described above.
  • the bioorthogonal functional group contained in the additional modification moiety may be the same as or different from the first bioorthogonal functional group and the second bioorthogonal functional group in (b) above, but different It is preferable.
  • the additional modifying moiety comprising a bioorthogonal functional group modifies an amino group in a side chain of a lysine residue present at one or more positions in the constant region of said two heavy chains.
  • the position of one or more (preferably 1 or 2, more preferably 1) lysine residues is at position 246/248, 288/290, or 317 of the human IgG heavy chain according to EU numbering.
  • the position where the additional modification moiety containing the bioorthogonal functional group is introduced includes the first modification moiety containing the first bioorthogonal functional group in (b) above, and the second bioorthogonal functional group.
  • the position is different from the position at which the second modification moiety is introduced.
  • the positions at which both the first modification moiety and the second modification moiety in (b) above are introduced are lysine residues at positions 246/248
  • the positions at which the additional modification moieties are introduced are: Lysine residues at positions 288/290 or 317 are preferred, and lysine residues at positions 288/290 are more preferred.
  • the positions at which both the first modification moiety and the second modification moiety are introduced in (b) above are lysine residues at positions 288/290
  • the positions at which the above additional modification moieties are introduced are 246/290.
  • Lysine residues at position 248 or 317 are preferred, and lysine residues at positions 246/248 are more preferred. If the position at which both the first modification moiety and the second modification moiety in (b) above are introduced is the lysine residue at position 317, the position at which the additional modification moiety is introduced is at position 246/248. , or lysine residues at positions 288/290.
  • an antibody derivative or its salt containing a bioorthogonal functional group depends on the specific raw material and the molecular weight of the product, but can be confirmed by, for example, electrophoresis, chromatography (e.g., gel filtration chromatography, ion This can be done by exchange chromatography, reversed phase column chromatography, HPLC), or mass spectrometry. Regioselectivity can be confirmed by peptide mapping as described above. The number of introduced bioorthogonal functional groups can be confirmed by mass spectrometry (DAR calculator (Agilent software) can be used in combination).
  • An antibody derivative containing a bioorthogonal functional group or a salt thereof can be appropriately purified by any method such as chromatography (eg, the above-mentioned chromatography and affinity chromatography).
  • Conjugate of an antibody and at least one functional substance or a salt thereof provides (a) a structural unit of an antibody (an immunoglobulin unit comprising two heavy chains and optionally two light chains), and ( b) contains a functional substance, and (c) the functional substance is introduced only into the constant region of one heavy chain in the immunoglobulin unit (i.e., in the constant region of one heavy chain in the immunoglobulin unit). (a functional substance is introduced into the constant region of the other heavy chain and no functional substance is introduced into the constant region of the other heavy chain), a conjugate of an antibody and a functional substance, or a salt thereof is provided. Definitions, examples, and preferred examples of antibodies, immunoglobulin units, conjugates of antibodies and functional substances, and constituent elements (eg, constant regions) are as described above.
  • the conjugate or a salt thereof may contain asparagine, glutamine, methionine, proline, serine, threonine, tryptophan, tyrosine, aspartic acid, glutamic acid, arginine, histidine, and lysine present in the constant region (preferably Fc region or CH2 domain).
  • Functional substances can be contained through modification of the functional groups in the side chains of any one or two or more (e.g., 2, 3, 4) of the 14 amino acid residues consisting of .
  • the conjugate or a salt thereof preferably comprises modification of a functional group in the side chain of any one of lysine, tyrosine, tryptophan, or cysteine present in the constant region (preferably Fc region or CH2 domain).
  • the immunoglobulin unit is modified by the corresponding modification via the functional group in the side chain of the above amino acid residue. It may have units regioselectively.
  • the conjugate comprises a constant region (preferably an Fc region or a CH2 domain) of one heavy chain in an antibody building block (an immunoglobulin unit comprising two heavy chains and optionally two light chains).
  • an immunoglobulin unit comprising two heavy chains and optionally two light chains.
  • the immunoglobulin unit can contain a functional substance (in other words, the immunoglobulin unit containing a functional substance through the amino group in the side chain of a lysine residue in the constant region of one heavy chain, and through the amino group in the side chain of a lysine residue in the constant region of the other heavy chain. (contains no functional substances). More specifically, the position of one or more (preferably 1 or 2, more preferably 1) lysine residues is at position 246/248, 288/290, or 317 of the human IgG heavy chain according to EU numbering.
  • the conjugate or salt thereof has the following formula (IVa): [During the ceremony, Ig refers to an immunoglobulin unit containing two heavy chains and optionally two light chains; L 1 indicates the first linker, Z represents a functional substance; The average modification percentage r of the immunoglobulin units by functional substances is 65-135%. ] or a salt thereof may be used.
  • the immunoglobulin unit denoted Ig, the first linker denoted L1 , the functional substance denoted Z, and the average modification percentage denoted r, as well as the definition, examples, and preferred examples of antibodies are as described above. be.
  • a particularly preferred bioorthogonal functional group is a thiol group.
  • the molecular weight of the partial structure represented by L 1 -Z may be 700 or less.
  • the conjugate or its salt has a very small ratio of the molecular weight of the partial structure to the overall molecular weight of the antibody, and therefore purification based on the difference in molecular weight is difficult.
  • the molecular weight of the partial structure represented by L 1 -Z is preferably 600 or less, more preferably 500 or less, even more preferably 400 or less, particularly preferably 300 or less, 250 or less, 200 or less, or It may be 100 or less.
  • the conjugate or salt thereof has the following formula (IVa-1): [During the ceremony, Ig refers to an immunoglobulin unit containing two heavy chains and optionally two light chains; W 1 represents an oxygen atom or a sulfur atom, L 3 indicates a third linker, Z represents a functional substance; The average modification percentage r of the immunoglobulin units by functional substances is 65-135%. ] or a salt thereof may be used. Immunoglobulin unit denoted by Ig, atom denoted by W 1 , third linker denoted by L 3 , functional substance denoted by Z, and average modification percentage denoted by r, as well as antibody definitions, examples, and preferences. Examples are given above.
  • the partial structure represented by L 3 may be (CH 2 ) n1 .
  • the definition, examples, and preferred examples of n1 are as described above.
  • the conjugate or salt thereof has the following formula (IVb):
  • Ig refers to an immunoglobulin unit containing two heavy chains and optionally two light chains; L 5 indicates the fifth linker, Z represents a functional substance; T 1 represents a monovalent group, The average modification percentage r of the immunoglobulin units by functional substances is 65-135%.
  • a particularly preferred bioorthogonal functional group is an azide group.
  • the molecular weight of the partial structure represented by L 5 (-Z)-T 1 may be 700 or less.
  • the molecular weight of the partial structure represented by L 5 (-Z)-T 1 is preferably 600 or less, more preferably 500 or less, even more preferably 400 or less, particularly preferably 300 or less, and 250 or less. , 200 or less, or 100 or less.
  • the conjugate or salt thereof has the following formula (IVb-1): [During the ceremony, Ig refers to an immunoglobulin unit containing two heavy chains and optionally two light chains; W 1 and W 2 each independently represent an oxygen atom or a sulfur atom, L 7 indicates the seventh linker, Z represents a functional substance; T 2 represents a monovalent group, The modification percentage r of the immunoglobulin unit by the functional substance is 65-135%. ] or a salt thereof may be used.
  • Immunoglobulin unit denoted by Ig
  • atoms denoted by W 1 and W 2
  • seventh linker denoted by L 7
  • functional substance denoted by Z
  • monovalent group denoted by T 2
  • r denoted by The average modification percentages, as well as antibody definitions, examples, and preferred examples are as described above.
  • a particularly preferred bioorthogonal functional group is an azide group.
  • the partial structure represented by L 7 may be (CH 2 ) n2 .
  • the definition, examples, and preferred examples of n2 are as described above.
  • the definition, examples, and preferred examples of n3 are as described above.
  • the method for producing a conjugate or a salt thereof is performed by reacting an antibody derivative containing a bioorthogonal functional group or a salt thereof with a functional substance to produce a conjugate or a salt thereof containing an antibody and a functional substance. It's okay.
  • the method for producing the conjugate or its salt may be performed by a method including the following (1) and (2): (1) producing an antibody derivative or a salt thereof containing a bioorthogonal functional group by the above method; and (2) reacting the antibody derivative or a salt thereof containing a bioorthogonal functional group with a functional substance; Producing a conjugate or a salt thereof comprising an antibody and a functional substance.
  • methods for producing the conjugate or its salt include, for example, the following (3-1) to (3-12) (FIGS. 2 to 6).
  • Method 3-1 An antibody derivative containing the structural unit represented by the above formula (IIIa) or a salt thereof is reacted with a functional substance to form a conjugate or A method for producing a conjugate of an antibody and a functional substance or a salt thereof, the method comprising producing a salt thereof.
  • Method 3-2 A method for producing a conjugate of an antibody and a functional substance or a salt thereof, including the following (1) and (2): (1) An antibody derivative or a salt thereof containing the structural unit represented by the above formula (IIIa) is obtained by cleaving an antibody or a salt thereof containing the structural unit represented by the above formula (IIIa) with a cleavage moiety. and (2) reacting an antibody derivative containing the structural unit represented by the above formula (IIIa) or a salt thereof with a functional substance to produce a conjugate containing the structural unit represented by the above formula (IVa). To produce gates or their salts.
  • a method for producing a conjugate of an antibody and a functional substance or a salt thereof including the following (1) to (3): (1) A compound represented by the above formula (Ia) or a salt thereof is reacted with an antibody or a salt thereof containing an immunoglobulin unit containing two heavy chains and optionally two light chains, and the above-mentioned producing an antibody or a salt thereof comprising a structural unit represented by formula (IIa); (2) An antibody derivative or a salt thereof containing the structural unit represented by the above formula (IIIa) is obtained by cleaving an antibody or a salt thereof containing the structural unit represented by the above formula (IIIa) with a cleavage moiety. and (3) reacting an antibody derivative containing the structural unit represented by the above formula (IIIa) or a salt thereof with a functional substance to produce a conjugate containing the structural unit represented by the above formula (IVa). To produce gates or their salts.
  • Method 3-4 An antibody derivative containing the structural unit represented by the above formula (IIIa-1) or a salt thereof is reacted with a functional substance to form the structural unit represented by the above formula (IVa-1).
  • a method for producing a conjugate of an antibody and a functional substance or a salt thereof the method comprising producing a conjugate or a salt thereof.
  • Method 3-5 A method for producing a conjugate of an antibody and a functional substance or a salt thereof, including the following (1) and (2): (1) Antibody derivatives containing the structural unit represented by the above formula (IIIa-1) obtained by cleaving an antibody containing the structural unit represented by the above formula (IIa-1) or a salt thereof with a cleavable moiety or a salt thereof; and (2) reacting an antibody derivative containing a structural unit represented by the above formula (IIIa-1) or a salt thereof with a functional substance to produce a salt thereof; Producing a conjugate or a salt thereof comprising the structural unit represented.
  • Method 3-6 A method for producing a conjugate of an antibody and a functional substance or a salt thereof, including the following (1) to (3): (1) A compound represented by the above formula (Ia-1) or a salt thereof is reacted with an antibody containing an immunoglobulin unit containing two heavy chains and optionally two light chains or a salt thereof.
  • a method for producing a conjugate of an antibody and a functional substance or a salt thereof including the following (1) and (2): (1) An antibody derivative or a salt thereof containing the structural unit represented by the above formula (IIIb) is obtained by cleaving an antibody or a salt thereof containing the structural unit represented by the above formula (IIIb) with a cleavage moiety. and (2) reacting an antibody derivative containing the structural unit represented by the above formula (IIIb) or a salt thereof with a functional substance to produce a conjugate containing the structural unit represented by the above formula (IVb). To produce gates or their salts.
  • a method for producing a conjugate of an antibody and a functional substance or a salt thereof including the following (1) to (3): (1) A compound represented by the above formula (Ib) or a salt thereof is reacted with an antibody or a salt thereof containing an immunoglobulin unit containing two heavy chains and optionally two light chains, and the above-mentioned producing an antibody or a salt thereof comprising a structural unit represented by formula (IIb); (2) An antibody derivative or a salt thereof containing the structural unit represented by the above formula (IIIb) is obtained by cleaving an antibody or a salt thereof containing the structural unit represented by the above formula (IIIb) with a cleavage moiety. and (3) reacting an antibody derivative containing the structural unit represented by the above formula (IIIb) or a salt thereof with a functional substance to produce a conjugate containing the structural unit represented by the above formula (IVb). To produce gates or their salts.
  • An antibody derivative containing a structural unit represented by the above formula (IIIb-1) or a salt thereof is reacted with a functional substance to form a structural unit represented by the above formula (IVb-1).
  • a method for producing a conjugate of an antibody and a functional substance or a salt thereof the method comprising producing a conjugate or a salt thereof.
  • a method for producing a conjugate of an antibody and a functional substance or a salt thereof including the following (1) and (2): (1) Antibody derivatives containing the structural unit represented by the above formula (IIIb-1) obtained by cleaving an antibody containing the structural unit represented by the above formula (IIIb-1) or a salt thereof with a cleavable moiety or a salt thereof; and (2) reacting an antibody derivative containing a structural unit represented by the above formula (IIIb-1) or a salt thereof with a functional substance to produce a salt thereof; Producing a conjugate or a salt thereof comprising the structural unit represented.
  • Method 3-12 A method for producing a conjugate of an antibody and a functional substance or a salt thereof, including the following (1) to (3): (1) A compound represented by the above formula (IB-1) or a salt thereof is reacted with an antibody containing an immunoglobulin unit containing two heavy chains and optionally two light chains or a salt thereof.
  • the above methods 3-1 to 3-12 may further include reacting the affinity substance of the present invention with a partial compound containing a reactive group for the antibody to produce the compound of the present invention or a salt thereof.
  • Good Figures 2-6).
  • the conjugate or its salt may further contain additional modifying moieties.
  • additional modifying moieties may be introduced into the heavy chain or light chain of the antibody, preferably in the heavy chain of the antibody (particularly in the constant region of the heavy chain).
  • the additional modification moiety may be an additional modification moiety that includes a functional substance.
  • the functional substances are the same as those described above.
  • the functional substance contained in the additional modification moiety may be the same as or different from the functional substance in (b) above, but is preferably different.
  • the additional modifying moiety comprising the functional agent is present through the modification of amino groups in the side chains of lysine residues present in one or more positions in the constant regions of said two heavy chains. , may be introduced into the constant regions of the two heavy chains.
  • the conjugate or a salt thereof can be used to bind the constant regions of two heavy chains (preferably the Fc region or the CH2 Additional modification moieties can be included through modification of the amino group in the side chain of one or more (preferably one or two, more preferably one) lysine residues in the domain).
  • the position of one or more (preferably 1 or 2, more preferably 1) lysine residues is at position 246/248, 288/290, or 317 of the human IgG heavy chain according to EU numbering.
  • the position where the additional modification moiety containing the functional substance is introduced is preferably different from the position where the functional substance described in (b) above is introduced.
  • the additional modification moiety is introduced into the lysine residue at positions 288/290 or 317. residues are preferred, and lysine residues at positions 288/290 are more preferred.
  • the additional modification moiety is introduced to the lysine residue at position 246/248 or 317. is preferred, and lysine residues at positions 246/248 are more preferred.
  • the additional modification moiety is introduced to the lysine residue at positions 246/248 or 288/290. is preferred.
  • Conjugate of an antibody and at least two functional substances or a salt thereof The present invention also provides an immunoconjugate comprising (a) two heavy chains consisting of a first and a second heavy chain and optionally two light chains. a globulin unit, and (b) a first modification moiety comprising a first functional substance and a second modification moiety comprising a second functional substance; (c) a first modification moiety is introduced into the constant region of the first heavy chain; (d) a second modification moiety is introduced into the constant region of the second heavy chain, and (e) an antibody and the first and second modifications, wherein the first and second modification moieties are different from each other; provides a conjugate of the moiety or a salt thereof.
  • Definitions, examples, and preferred examples of antibodies, immunoglobulin units, and bioorthogonal functional groups, and their constituent elements (eg, constant regions) are as described above.
  • the conjugate or a salt thereof may contain asparagine, glutamine, methionine, proline, serine, threonine, tryptophan, tyrosine, aspartic acid, glutamic acid, arginine, histidine, and lysine present in the constant region (preferably Fc region or CH2 domain).
  • the antibody derivative or a salt thereof is preferably modified with a functional group in the side chain of any one of lysine, tyrosine, tryptophan, or cysteine present in the constant region (preferably Fc region or CH2 domain). more preferably via modification of a functional group in the side chain of any one of the amino acids lysine, tyrosine, or tryptophan, even more preferably via modification of a functional group in the side chain of lysine or tyrosine.
  • a first modification moiety comprising a first functional substance
  • a second modification comprising a second functional substance, through modification, particularly preferably through modification of an amino group in the side chain of lysine. can contain parts.
  • the positions of these amino acid residues in the constant region are as described above.
  • the modification position of an antibody or its salt with a functional substance can be confirmed by peptide mapping. Modifications may be regioselective, as described above. Therefore, in the below-mentioned formula, the immunoglobulin unit may have a corresponding modification unit regioselectively via the functional group in the side chain of the amino acid residue.
  • the conjugate or salt thereof binds to the constant regions of the first and second heavy chains (in an immunoglobulin unit comprising two heavy chains and optionally two light chains) of an antibody (an immunoglobulin unit comprising two heavy chains and optionally two light chains).
  • the first functional substance through modification of the amino group in the side chain of one or more (preferably 1 or 2, more preferably 1) lysine residues in the Fc region or CH2 domain).
  • a first modification moiety comprising a first modification moiety
  • a second modification moiety comprising a second functional substance. More specifically, the position of one or more (preferably 1 or 2, more preferably 1) lysine residues is at position 246/248, 288/290, or 317 of the human IgG heavy chain according to EU numbering.
  • the immunoglobulin unit may have a corresponding modification unit regioselectively via the amino group in the side chain of the lysine residue.
  • the conjugate or salt thereof has the following formula (VIIa): [During the ceremony, Ig indicates the immunoglobulin unit, L L1 and L R1 each independently represent a first linker, ZL represents the first functional substance, Z R represents a second functional substance, The average percentage modification r L of said immunoglobulin units by the first modifying moiety and the average percentage modification r R of said immunoglobulin units by the second modifying moiety are each between 65 and 135%. ] or a salt thereof may be used.
  • a conjugate containing a structural unit represented by formula (VIIa) or a salt thereof can be produced by reacting an antibody derivative containing a structural unit represented by formula (VIIa) or a salt thereof with a functional substance.
  • a functional substance e.g., a functional substance that can be produced by reacting an antibody derivative containing a structural unit represented by formula (VIIa) or a salt thereof with a functional substance.
  • the reaction can be carried out in one reaction.
  • the reaction can be a single reaction (e.g., if the two different bioorthogonal functional groups can react under similar reaction conditions) or a double reaction (e.g., (when two different bioorthogonal functional groups can react under different reaction conditions). Details of the reaction are as described above.
  • the antibody derivative or salt thereof has the following formula (VIIa-1): [During the ceremony, Ig indicates the immunoglobulin unit, W L1 and W R1 each independently represent an oxygen atom or a sulfur atom, L L3 and L R3 each independently represent a third linker, ZL represents the first functional substance, Z R represents a second functional substance, The average percentage modification r L of said immunoglobulin units by the first modifying moiety and the average percentage modification r R of said immunoglobulin units by the second modifying moiety are each between 65 and 135%. ] or a salt thereof may be used.
  • the conjugate containing the structural unit represented by formula (VIIa-1) or a salt thereof can be produced by reacting a functional substance with an antibody derivative containing the structural unit represented by formula (VIIa-1) or a salt thereof. This can be done by The cleavage reaction can be carried out in one reaction. Details of the reaction are as described above.
  • the conjugate or salt thereof has the following formula (VIIb): [During the ceremony, Ig indicates the immunoglobulin unit, L L5 and L R5 each independently represent a fifth linker, ZL represents the first functional substance, Z R represents a second functional substance, T L1 and T R1 each independently represent a monovalent group, The average percentage modification r L of said immunoglobulin units by the first modifying moiety and the average percentage modification r R of said immunoglobulin units by the second modifying moiety are each between 65 and 135%. ] or a salt thereof may be used.
  • the conjugate containing the structural unit represented by formula (VIIb) or a salt thereof can be produced by reacting an antibody derivative containing the structural unit represented by formula (VIIb) or a salt thereof with a functional substance. can.
  • the reaction can be carried out in one reaction.
  • the reaction can be a single reaction (e.g., if the two different bioorthogonal functional groups can react under similar reaction conditions) or a double reaction (e.g., (when two different bioorthogonal functional groups can react under different reaction conditions). Details of the reaction are as described above.
  • the conjugate or salt thereof has the following formula (VIIb-1): [During the ceremony, Ig indicates the immunoglobulin unit, W L1 and W L2 and W R1 and W R2 each independently represent an oxygen atom or a sulfur atom, L L7 and L R7 each independently represent a seventh linker, ZL represents the first functional substance, Z R represents a second functional substance, T L2 and T R2 each independently represent a monovalent group, The average percentage modification r L of said immunoglobulin units by the first modifying moiety and the average percentage modification r R of said immunoglobulin units by the second modifying moiety are each between 65 and 135%. ] or a salt thereof may be used.
  • the conjugate containing the structural unit represented by formula (VIIb-1) or a salt thereof can be produced by reacting a functional substance with an antibody derivative containing the structural unit represented by formula (VIIb-1) or a salt thereof. This can be done by When two bioorthogonal functional groups in the antibody derivative containing the structural unit represented by formula (VIb-1) or a salt thereof are the same, the reaction can be carried out in one reaction. When the two bioorthogonal functional groups are different, the reaction can be a single reaction (e.g., if the two different bioorthogonal functional groups can react under similar reaction conditions) or a double reaction (e.g., (when two different bioorthogonal functional groups can react under different reaction conditions). Details of the reaction are as described above.
  • the conjugate or salt thereof has the following formula (VIIc): [During the ceremony, Ig indicates the immunoglobulin unit, L R1 indicates the first linker, L L5 indicates the fifth linker, ZL represents the first functional substance, Z R represents a second functional substance, T L1 represents a monovalent group, The average percentage modification r L of said immunoglobulin units by the first modifying moiety and the average percentage modification r R of said immunoglobulin units by the second modifying moiety are each between 65 and 135%. ] or a salt thereof may be used.
  • the conjugate containing the structural unit represented by formula (VIIc) or a salt thereof can be produced by reacting a functional substance with an antibody derivative containing the structural unit represented by formula (VIIc) or a salt thereof. can.
  • the reaction can be carried out in one reaction.
  • the reaction can be a single reaction (e.g., if the two different bioorthogonal functional groups can react under similar reaction conditions) or a double reaction (e.g., (when two different bioorthogonal functional groups can react under different reaction conditions). Details of the reaction are as described above.
  • the conjugate or salt thereof has the following formula (VIIc-1): [During the ceremony, Ig indicates the immunoglobulin unit, W L1 and W L2 and W R1 each independently represent an oxygen atom or a sulfur atom, L R3 represents a third linker, L L7 indicates the seventh linker, ZL represents the first functional substance, Z R represents a second functional substance, T L2 represents a monovalent group, The average percentage modification r L of said immunoglobulin units by the first modifying moiety and the average percentage modification r R of said immunoglobulin units by the second modifying moiety are each between 65 and 135%. ] or a salt thereof may be used.
  • the conjugate containing the structural unit represented by formula (VIIc-1) or a salt thereof can be produced by reacting a functional substance with an antibody derivative containing the structural unit represented by formula (VIIc-1) or a salt thereof. This can be done by When the two bioorthogonal functional groups in the antibody derivative or salt thereof containing the structural unit represented by formula (VIc) are the same, the reaction can be carried out in one reaction. When the two bioorthogonal functional groups are different, the reaction can be a single reaction (e.g., if the two different bioorthogonal functional groups can react under similar reaction conditions) or a double reaction (e.g., (when two different bioorthogonal functional groups can react under different reaction conditions). Details of the reaction are as described above.
  • the third linkers represented by L L3 and L R3 may be the same or different, and are preferably different.
  • the fifth linker denoted L L5 and L R5 is similar to the fifth linker denoted L 5 .
  • the first linkers represented by L L5 and L R5 may be the same or different, and are preferably different.
  • the seventh linker designated L L7 and L R7 is similar to the seventh linker designated L 7 .
  • the seventh linkers represented by L L7 and L R7 may be the same or different, and are preferably different.
  • the first group containing the first bioorthogonal functional group represented by B L and the second group containing the second bioorthogonal functional group represented by B R are the bioorthogonal functional group represented by B.
  • the first group containing the first bioorthogonal functional group represented by B L and the second group containing the second bioorthogonal functional group represented by B R may be the same or different. Preferably, they are different.
  • the monovalent groups represented by T L1 and T R1 are the same as the monovalent group represented by T 1 .
  • the monovalent groups represented by T L1 and T R1 may be the same or different.
  • the monovalent groups represented by T L2 and T R2 are the same as the monovalent group represented by T 2 .
  • the monovalent groups represented by T L2 and T R2 may be the same or different.
  • the degree of average modification percentage denoted by r L and r R and the method for determining it are the same as the average modification percentage denoted by r.
  • the average modification percentages represented by r L and r R may be the same or different, and are preferably different.
  • the molecular weight of the substructure represented by L L1 -Z L and/or L R1 -Z R is the same as that represented by L 1 -Z in formula (IVa).
  • the molecular weight may be the same as the above molecular weight of the partial structure.
  • the third linker represented by L L3 and/or L R3 is the third linker represented by L 3 in formula (IVa-1). It may be the same as the linker (CH 2 ) n1 .
  • the monovalent group represented by T L1 , T R1 , T L2 , and/or T R2 is , T 1 and/or T 2 in formula (IVb), which may be an optionally substituted hydroxyamino group.
  • the molecular weight of the substructure represented by L L5 (-Z L )-T L1 and/or L R5 (-Z R )-T R1 is The molecular weight may be the same as that of the partial structure represented by L 5 (-Z)-T 1 in (IVb).
  • the seventh linker represented by L L7 and/or L R7 is the seventh linker represented by L 7 in formula (IVb-1). It may be the same as the linker (CH 2 ) n2 .
  • the conjugate or its salt may further contain additional modifying moieties.
  • additional modifying moieties may be introduced into the heavy chain or light chain of the antibody, preferably in the heavy chain of the antibody (particularly in the constant region of the heavy chain).
  • the additional modification moiety may be an additional modification moiety that includes a functional substance.
  • the functional substances are the same as those described above.
  • the functional substance contained in the additional modification moiety may be the same as or different from the first functional substance and the second functional substance in (b) above, but it is preferable that they are different.
  • the additional modifying moiety comprising the functional agent is present through the modification of amino groups in the side chains of lysine residues present in one or more positions in the constant regions of said two heavy chains. , may be introduced into the constant regions of the two heavy chains.
  • the conjugate or a salt thereof can be used to bind the constant regions of two heavy chains (preferably the Fc region or the CH2 Additional modification moieties can be included through modification of the amino group in the side chain of one or more (preferably one or two, more preferably one) lysine residues in the domain).
  • the position of one or more (preferably 1 or 2, more preferably 1) lysine residues is at position 246/248, 288/290, or 317 of the human IgG heavy chain according to EU numbering.
  • the position where the additional modification moiety containing the functional substance is introduced is the first modification moiety containing the first functional substance and the second modification moiety containing the second functional substance in (b) above. It is preferable that the position is different from the position where it is introduced.
  • the positions at which both the first modification moiety and the second modification moiety in (b) above are introduced are lysine residues at positions 246/248
  • the positions at which the additional modification moieties are introduced are: Lysine residues at positions 288/290 or 317 are preferred, and lysine residues at positions 288/290 are more preferred.
  • the positions at which the above additional modification moieties are introduced are 246/290. Lysine residues at position 248 or 317 are preferred, and lysine residues at positions 246/248 are more preferred.
  • the position at which both the first modification moiety and the second modification moiety in (b) above are introduced is the lysine residue at position 317
  • the position at which the additional modification moiety is introduced is at position 246/248. , or lysine residues at positions 288/290.
  • Confirmation of the formation of the conjugate or its salt depends on the specific raw materials and the molecular weight of the product, but can be confirmed by, for example, electrophoresis, chromatography (e.g., gel filtration chromatography, ion exchange chromatography, reversed phase column This can be carried out by chromatography, HPLC) or mass spectrometry. Regioselectivity can be confirmed by peptide mapping as described above. The number of introduced functional substances can be confirmed by mass spectrometry (DAR calculator (Agilent software) can be used in combination).
  • the conjugate or a salt thereof can be appropriately purified by any method such as chromatography (eg, the above-mentioned chromatography and affinity chromatography).
  • the compound of the present invention or a salt thereof can easily modify only one heavy chain in an antibody constitutional unit (immunoglobulin unit containing two heavy chains and, if necessary, two light chains). (The average modification percentage r of immunoglobulin units is 65-135%).
  • the compounds of the present invention or salts thereof can also regioselectively modify specific amino acid residues (preferably lysine residues) in the heavy chain of the immunoglobulin unit. Therefore, the present invention provides a reagent for antibody derivatization containing the compound of the present invention or a salt thereof.
  • the reagent of the present invention may be provided in the form of a composition that further contains other components.
  • Such other components include, for example, solutions, stabilizers (eg, antioxidants, preservatives).
  • an aqueous solution is preferred.
  • aqueous solutions include water (e.g., distilled water, sterile distilled water, purified water, physiological saline), buffer solutions (e.g., phosphoric acid aqueous solution, Tris-hydrochloric acid buffer, carbonic acid-bicarbonate buffer, boric acid aqueous solution). , glycine-sodium hydroxide buffer, citrate buffer), but buffers are preferred.
  • the pH of the solution is, for example, 5.0 to 9.0, preferably 5.5 to 8.5.
  • Reagents of the invention can be provided in liquid or powder form (eg, lyophilized powder).
  • the affinity substance-modified antibody, antibody derivative, or salt thereof of the present invention is useful, for example, as an intermediate for the preparation of the conjugate of the present invention or a salt thereof.
  • the conjugate of the present invention or a salt thereof is useful, for example, as a medicine or a reagent (eg, a diagnostic agent, a research reagent).
  • a medicine or a reagent eg, a diagnostic agent, a research reagent.
  • the present invention in which only one heavy chain in the antibody structural unit is modified (the average modification percentage r of immunoglobulin units is 65 to 135%), and moreover, it is regioselectively modified with a functional substance.
  • Conjugates of or salts thereof are useful as pharmaceuticals. It has been reported that changing the number and binding position of drugs in an antibody-drug conjugate (ADC) changes the pharmacokinetics, drug release rate, and effect. For these reasons, next-generation ADCs are required to control the number and position of conjugated drugs. It is believed that if the number and position are constant, the problems of expected efficiency, variations in conjugated drugs, and lot differences, so-called regulation, will be solved. Therefore, the conjugate of the present
  • the conjugate of the present invention or a salt thereof may be provided in the form of a pharmaceutical composition.
  • Such pharmaceutical compositions may contain, in addition to the conjugate of the present invention or a salt thereof, a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers include, for example, excipients such as sucrose, starch, mannitol, sorbitol, lactose, glucose, cellulose, talc, calcium phosphate, calcium carbonate, cellulose, methylcellulose, hydroxypropylcellulose, polypropylpyrrolidone.
  • binders such as gelatin, gum arabic, polyethylene glycol, sucrose, starch, starch, carboxymethyl cellulose, hydroxypropyl starch, disintegrants such as sodium bicarbonate, calcium phosphate, calcium citrate, magnesium stearate, aerosil, talc, lauryl.
  • disintegrants such as sodium bicarbonate, calcium phosphate, calcium citrate, magnesium stearate, aerosil, talc, lauryl.
  • Lubricants such as sodium sulfate, citric acid, menthol, glycyrrhizin ammonium salt, glycine, fragrances such as orange powder, preservatives such as sodium benzoate, sodium bisulfite, methylparaben, propylparaben, citric acid, sodium citrate, acetic acid.
  • stabilizers such as methylcellulose, polyvinylpyrrolidone, aluminum stearate, suspending agents such as surfactants, diluents such as water, physiological saline, orange juice, cocoa butter, polyethylene glycol, white kerosene, etc. Examples include, but are not limited to, base wax.
  • the conjugates of the invention or salts thereof may also have any modifications that provide stability (eg, PEGylation).
  • Formulations suitable for oral administration include solutions in which an effective amount of the ligand is dissolved in a diluent such as water, physiological saline, or orange juice, capsules, sachets, or tablets containing an effective amount of the ligand in the form of solids or granules.
  • a diluent such as water, physiological saline, or orange juice
  • a diluent such as water, physiological saline, or orange juice
  • capsules, sachets or tablets containing an effective amount of the ligand in the form of solids or granules.
  • These include tablets, suspensions in which an effective amount of the active ingredient is suspended in an appropriate dispersion medium, and emulsions in which a solution in which an effective amount of the active ingredient is dissolved is dispersed and emulsified in an appropriate
  • compositions suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions containing antioxidants, buffers, bacteriostatic agents, and tonicity agents. etc. may be included. Also included are aqueous and non-aqueous sterile suspensions, which may contain suspending agents, solubilizers, thickeners, stabilizers, preservatives, and the like.
  • the dosage of the pharmaceutical composition varies depending on the type and activity of the active ingredient, the severity of the disease, the species of animal to be administered, the drug acceptability of the subject, body weight, age, etc., and can be set as appropriate.
  • the present invention in order to be able to modify the immunoglobulin unit, it is preferable to bind a compound having a reactive group to the immunoglobulin unit to the affinity substance. Furthermore, in order to remove the affinity substance after modification of the immunoglobulin unit, it is preferable to include a cleavable moiety between the reactive group and the affinity substance. Furthermore, in order to achieve specific binding of the compound to a specific site in the affinity substance, it is preferable to design the affinity substance so that there is only one site capable of reacting with the compound.
  • an affinity polypeptide is designed such that only one group is present in the affinity polypeptide. Therefore, an affinity polypeptide was designed as follows. B) The polypeptide contains only one K residue. C) The N-terminal amino acid is Q (glutamine), which causes pyroglutamylation and converts the N-terminal amino group into an amide.
  • polypeptide affinity substances were designed as shown in (a) to (h) below.
  • affinity polypeptides As the affinity polypeptides, QET-Z34CM-PA32-Fc3K, QET-Z34CM-PA48-Fc3K, QET-Fc3K-PA32-Z34CM, QET-Fc3K-PA48-Z34CM, QET-Fc3K-PA32-ProAR, QET-Fc 3K - Eight types of amino acid sequences, PA48-ProAR, QET-ProAR-PA32-Z34CK, and QET-ProAR-PA48-Z34CK, were designed, and the base sequences encoding these polypeptides were converted to C.I. It was designed taking into consideration the codon usage frequency of C. glutamicum. Furthermore, C. The following expression cassettes were designed to enable secretory expression by C. glutamicum.
  • QET-Z34CM-PA32-Fc3K is C. glutamicum ATCC13869 strain, 30 amino acid residues of the CspB signal peptide, QET, the N-terminal 3 amino acid residues of the CspB mature protein derived from the same strain, and a fusion protein of Z34CM-PA32-Fc3K (hereinafter referred to as "CspBss-QET-Z34CM-PA32"). -Fc3K”) was secreted and expressed.
  • the nucleotide sequence and amino acid sequence encoding the designed CspBss-QET-Z34CM-PA32-Fc3K are shown in SEQ ID NOs: 9 and 10, respectively.
  • QET-Z34CM-PA48-Fc3K is C. glutamicum ATCC 13869 strain, the N-terminal 3 amino acid residues QET of the CspB mature protein derived from the same strain, and the Z34CM-PA48-Fc3K fusion protein (hereinafter referred to as "CspBss-QET-Z34CM-PA48"). -Fc3K”) was secreted and expressed.
  • the nucleotide sequence and amino acid sequence encoding the designed CspBss-QET-Z34CM-PA48-Fc3K are shown in SEQ ID NOs: 11 and 12, respectively.
  • QET-Fc3K-PA32-Z34CM is C. glutamicum ATCC 13869 strain, the N-terminal 3 amino acid residues QET of the CspB mature protein derived from the same strain, and a fusion protein of Fc3K-PA32-Z34CM (hereinafter referred to as "CspBss-QET-Fc3K-PA32"). -Z34CM”) was secreted and expressed.
  • the nucleotide sequence and amino acid sequence encoding the designed CspBss-QET-Fc3K-PA32-Z34CM are shown in SEQ ID NOs: 13 and 14, respectively.
  • QET-Fc3K-PA48-Z34CM is C. glutamicum ATCC 13869 strain, the N-terminal 3 amino acid residues QET of the CspB mature protein derived from the same strain, and a fusion protein of Fc3K-PA48-Z34CM (hereinafter referred to as "CspBss-QET-Fc3K-PA48"). -Z34CM”) was secreted and expressed.
  • the nucleotide sequence and amino acid sequence encoding the designed CspBss-QET-Fc3K-PA48-Z34CM are shown in SEQ ID NOs: 15 and 16, respectively.
  • QET-Fc3K-PA32-ProAR is a C. glutamicum ATCC 13869 strain, the N-terminal 3 amino acid residues QET of the CspB mature protein derived from the same strain, and the Fc3K-PA32-ProAR fusion protein (hereinafter referred to as "CspBss-QET-Fc3K-PA32").
  • CspBss-QET-Fc3K-PA32 Fc3K-PA32-ProAR fusion protein
  • QET-Fc3K-PA48-ProAR is a C. glutamicum ATCC 13869 strain, the N-terminal 3 amino acid residues QET of the CspB mature protein derived from the same strain, and the Fc3K-PA48-ProAR fusion protein (hereinafter referred to as "CspBss-QET-Fc3K-PA48").
  • CspBss-QET-Fc3K-PA48 Fc3K-PA48-ProAR fusion protein
  • QET-ProAR-PA32-Z34CK is C. glutamicum ATCC 13869 strain, the N-terminal 3 amino acid residues QET of the CspB mature protein derived from the same strain, and a fusion protein of ProAR-PA32-Z34CK (hereinafter referred to as "CspBss-QET-ProAR-PA32"). -Z34CK”) was secreted and expressed.
  • the nucleotide sequence and amino acid sequence encoding the designed CspBss-QET-ProAR-PA32-Z34CK are shown in SEQ ID NOs: 21 and 22, respectively.
  • QET-ProAR-PA48-Z34CK is C. glutamicum ATCC 13869 strain, the N-terminal 3 amino acid residues QET of the CspB mature protein derived from the same strain, and a fusion protein of ProAR-PA48-Z34CK (hereinafter referred to as "CspBss-QET-ProAR-PA48"). -Z34CK”) was secreted and expressed.
  • the nucleotide sequence and amino acid sequence encoding the designed CspBss-QET-ProAR-PA48-Z34CK are shown in SEQ ID NOs: 23 and 24, respectively.
  • An expression cassette for eight types of affinity polypeptides was designed and completely synthesized, in which the promoter of the cspB gene derived from the ATCC 13869 strain of C. glutamicum was connected, and a KpnI site was added to the 5'-side and a BamHI site was added to the 3'-side.
  • pPK4_CspBss-QET- a secretory expression plasmid for affinity polypeptide
  • Each of the obtained transformants was cultured in an MMTG liquid medium containing 25 mg/L of kanamycin (120 g of glucose, 3 g of magnesium sulfate heptahydrate, 30 g of ammonium sulfate, 1.5 g of potassium dihydrogen phosphate, 0 g of iron sulfate heptahydrate).
  • kanamycin 120 g of glucose, 3 g of magnesium sulfate heptahydrate, 30 g of ammonium sulfate, 1.5 g of potassium dihydrogen phosphate, 0 g of iron sulfate heptahydrate.
  • a polypeptide band presumed to be Z34CM ( Figure 16, Lanes 14-17), a polypeptide band presumed to be QET-Fc3K-PA32-ProAR in the culture supernatant of YDK0107/pPK4_CspBss-QET-Fc3K-PA32-ProAR strain ( Figure 16, Lanes 18-21), a polypeptide band presumed to be QET-Fc3K-PA48-ProAR in the culture supernatant of YDK0107/pPK4_CspBss-QET-Fc3K-PA48-ProAR strain ( Figure 16, Lanes 22-25), YDK0107 A polypeptide band estimated to be QET-ProAR-PA32-Z34CK ( Figure 16, Lanes 26-29) in the culture supernatant of the /pPK4_CspBss-QET-ProAR-PA32-Z34CK strain, and YDK0107/pPK4_CspBss-QET
  • Example 2 Purification of affinity substance and measurement of affinity between affinity substance and IgG1 antibody Fc region Culture in a fermentation layer for mass culturing the strain obtained in Example 1 (1-5) was carried out according to a known method (Appl Microbiol Biotechnol (2008) 78:621-625). The centrifuged supernatant of the culture solution was subjected to sterile filtration using a 0.2 ⁇ m PVDF membrane filter. In order to remove contaminant components in the culture solution, the expressed polypeptide was purified by preparative HPLC using a cation exchange column. Affinity measurements of the resulting polypeptides were subsequently performed. Specifically, it was carried out as follows.
  • the affinity between each polypeptide and the IgG1 antibody Fc region was evaluated by SPR measurement (Biacore T200 (GE Healthcare)). The measurement was performed at 25°C, and HBS-EP+ was used as the running buffer.
  • human IgG1 antibody Fc protein was diluted to 30 ⁇ g/mL with sodium acetate buffer pH 4.5 (GE Healthcare), and about 1400 RU was immobilized on the surface of a CM5 Sensor Chip (GE Healthcare) using the amine coupling method. Each peptide was diluted to 100 nM with HBS-EP+ buffer containing 0.1% DMSO.
  • the measurement was performed using a single cycle kinetics method, an analyte addition time of 120 seconds, a flow rate of 30 ⁇ L/min, and a dissociation time of 120 seconds. Further, the obtained sensorgram was fitted by affinity, and the value of the dissociation constant (KD) was calculated. As a result of the measurement, it was confirmed that all the expressed polypeptides bound to the human IgG1 antibody Fc protein (Table 1 and Figures 17-1 to 17-8).
  • Example 3 Preparation of a compound having an affinity substance for an antibody, a cleavable moiety, and a reactive group A compound having a cleavable moiety and a reactive group was bound to a purified polypeptide.
  • affinity reagent (1) was prepared by amidating the affinity substance QET-Z34CM-PA32-Fc3K prepared in Example 1-2.
  • affinity reagent (2) was prepared from the affinity substance QET-Z34CM-PA48-Fc3K prepared in Example 1-2.
  • affinity reagent (3) was prepared from the affinity substance QET-Fc3K-PA32-Z34CM prepared in Example 1-2.
  • affinity reagent (4) was prepared from the affinity substance QET-Fc3K-PA48-Z34CM prepared in Example 1-2.
  • an affinity reagent (5) was prepared from the affinity substance QET-Fc3K-PA32-ProAR prepared in Example 1-2.
  • an affinity reagent (6) was prepared from the affinity substance QET-Fc3K-PA48-ProAR prepared in Example 1-2.
  • an affinity reagent (7) was prepared from the affinity substance QET-ProAR-PA32-Z34CK prepared in Example 1-2.
  • an affinity reagent (8) was prepared from the affinity substance QET-ProAR-PA48-Z34CK prepared in Example 1-2.
  • Example 4 Specific modification of anti-HER2 IgG antibody trastuzumab and synthesis of ADC mimic (4-1) Specific modification of anti-HER2 antibody trastuzumab using affinity reagent (4)
  • Anti-HER2 IgG antibody trastuzumab (Chugai Pharmaceutical) 500 ⁇ g was dissolved in 171 ⁇ L of 50 mM sodium acetate buffer (pH 5.5).
  • the mass was measured by ESI-TOFMS, and a peak of the raw material trastuzumab was observed at 148400, indicating that it was a product with one binding peptide introduced. A peak was observed at 159300, and a peak at 170390 was observed for the product in which two binding peptides were introduced.
  • the antibody obtained above was subjected to a thioester group cleavage reaction according to a previous report (WO2019/0240287) to obtain a thiol group-introduced antibody derivative (T-1-SH) into which one thiol group was introduced.
  • T-1-SH thiol group-introduced antibody derivative
  • pyroglutamylation of glutamic acid residue (-18.011 Da) was set as a dynamic modification to the N-terminus of the protein
  • lysine addition (+128.095 Da) was set as a dynamic modification to the C-terminus of the protein.
  • filters were set to include only those with Confidence Score of 80 or higher, Mass Accuracy at the time of peptide identification within 5 ppm, and MS/MS observed.
  • residue numbers of lysine residues on the heavy chain VH domain and light chain, the numbers in the sequence (i.e., the N-terminal amino acid is the first. The same applies hereinafter) are used for heavy chain CH1, CH2, CH3. Domains are expressed using EU numbering.
  • Example 5 Specific modification of other IgG antibodies Various antibodies were reacted with the modification reagent (4) according to the conditions of Example (4-1). ESI-TOFMS was measured in the same manner as in Example 4-1, and the average peptide/antibody binding ratio was calculated using a DAR calculator. A list of antibodies and average peptide/antibody binding ratios are listed in Table 4.
  • Example 6 Specific introduction of an azide group into the anti-HER2 IgG antibody trastuzumab and synthesis of ADC mimic (6-1) Synthesis of a low molecular weight linker having an azide group (6-1-1) Linker intermediate (11) ) synthesis
  • 5-azidopentanoic acid 800 mg, 5.59 mmol was dissolved in THF (14 mL), and isobutyl chloroformate (808 ⁇ L, 6.15 mmol) and N-methylmorpholine (873 ⁇ L, 8.39 mmol) were added to the mixture at 0°C for 30 minutes. After stirring for several minutes, hydrazine hydrate (1.36 g, 6.71 mmol) dissolved in 1M NaOH aqueous solution (4 mL) was added, and the mixture was stirred at room temperature for 3 hours.
  • Linker intermediate (12) (2.20 g, 5.23 mmol) was dissolved in dichloromethane (10 mL), trifluoroacetic acid (10 mL) was added, and after stirring at room temperature for 1 hour, it was concentrated under reduced pressure to remove dichloromethane. By adding water and freeze-drying, linker intermediate (13) was obtained (1.98 g, 5.43 mmo).
  • Linker intermediate (13) (100 mg, 0.274 mmol) was dissolved in dichloromethane (3 mL), (40.6 ⁇ L, 0.280 mmol), benzotriazol-1-yloxy (150 mg, 0.288 mmol), DIPEA (70. 1 ⁇ L, 0.412 mmol) was added thereto, and the mixture was stirred at room temperature for 2 hours. A 1M HCl aqueous solution was added to adjust the pH of the system to 3, dichloromethane was added to dilute the system, and after washing with water and brine, sodium sulfate was added.
  • linker intermediate (14) (84.7 mg, 0.171 mmol).
  • affinity reagent (9) was prepared by amidation of affinity substance QET-Fc3K-PA48-Z34CM and linker intermediate (16).
  • Example 7 Synthesis of an antibody into which one molecule of a thiol group and an azide group are introduced (7-1) Synthesis of Trastuzumab (T-2-N3/SH) into which one molecule of a thiol group and an azide group are introduced ( 7-1-1) Single molecule-specific modification of anti-HER2 antibody trastuzumab affinity reagent by conjugation and HPLC purification
  • Example 7-1-1 the antibody into which one molecule of the peptide reagent obtained in Example 7-1-1 was introduced was tested as previously reported (WO2019/240287A1).
  • Conjugation was performed according to the method of As a result, antibodies into which modification reagents (17) and (18) were introduced were obtained.
  • DAR analysis of antibodies introduced with peptide reagents (17) and (18) was performed by HIC-HPLC analysis according to a previous report (Anal.Chem., 2019, 91, 20, 12724-12732). I confirmed that it was done.
  • a methoxyamine solution was added to the antibody introduced with the modification reagents (17) and (18) obtained in Example 7-1-2, referring to the method previously reported (WO2019/240287A1), and the mixture was incubated at room temperature for 3 hours. The cleavage reaction was performed by shaking. As a result, an antibody (T-2-N3/SH) into which one thiol group and one azido group were introduced was obtained. DAR analysis of the antibody (T-2-N3/SH) into which a thiol group and an azide group were introduced one molecule at a time was performed using HIC-HPLC analysis according to a previous report (Anal. Chem., 2019, 91, 20, 12724-12732). It was confirmed that the thiol group and the azide group were introduced one molecule at a time.
  • Immobilized Pepsin (Thermo Fisher) was added to a 10 mg/mL acetate buffer (50 mM Sodium acetate, pH 4.5) solution of the anti-human EGFR monoclonal antibody Cetuximab (Merck Biopharma), and the mixture was shaken at 37°C for 4 hours. After the reaction solution was purified using a NAP-25 desalting column (manufactured by Cytiva), two F(ab) molecules of Cetuximab were obtained using AKTA pure25 (manufactured by Cytiva). Fab was analyzed by ESI-TOFMS according to a previous report (Anal. Chem., 2019, 91, 20, 12724-12732), and a peak was observed at 101581.
  • a PBSE buffer solution (10 mM) of 2 -Mercaptoethylamine was added to the PBSE buffer solution (10 mM Phosphate Buffered Saline (PBS), 10 mM EDTA, pH 7.4) of F(ab) 2 obtained in Example 8-1-1. was added and shaken at 37°C for 1 hour.
  • the reaction solution was purified using a NAP-25 desalting column (manufactured by Cytiva) to obtain Fab.
  • Fab analysis was performed by ESI-TOFMS analysis according to a previous report (Anal. Chem., 2019, 91, 20, 12724-12732), and a peak was observed at 50793.
  • Example 8-1-3 a PEG12-DBCO group-introduced Fab was obtained by using the Cetuximab Fab obtained in Example 8-1-2 and commercially available DBCO-PEG12-Maleimide.
  • ESI-TOFMS analysis was performed according to a previous report (Anal. Chem., 2019, 91, 20, 12724-12732), and a peak was observed at 52846.
  • a dimethylformamide solution (for the Fab) of commercially available DBCO-PEG4-Maleimide was added to an acetate buffer (50 mM Sodium acetate, pH 5.5) solution of the Fab of Trastuzumab into which the azide group obtained in Example 8-2-4 was introduced. 3 equivalents of dimethylformamide (DMF) (8% v/v) solution) was added and shaken for 20 hours.
  • the reaction solution was purified using a NAP-25 desalting column (manufactured by Cytiva) to synthesize a Fab of Tastuzumab into which a maleimide group had been introduced.
  • ESI-TOFMS analysis was performed according to a previous report (Anal. Chem., 2019, 91, 20, 12724-12732), and a peak was observed at 50733.
  • Example 8-2 was added to a PBSE buffer (10 mM Phosphate Buffered Saline (PBS), 10 mM EDTA, pH 7.4) solution of Cetuximab into which one molecule of the thiol group and azide group synthesized in Example 7-2 was introduced.
  • a PBSE buffer (10 mM Phosphate Buffered Saline (PBS), 10 mM EDTA, pH 7.4) solution (1.5 equivalents to the antibody) of the Trastuzumab Fab into which the maleimide group synthesized in step-5 was added. and shaken for 1 hour.
  • Tri-specific antibody (M-1) was obtained by purifying the reaction solution using AKTA pure25 (manufactured by Cytiva). Tri-specific antibody (M-1) was analyzed by ESI-TOFMS analysis according to a previous report (Anal. Chem., 2019, 91, 20, 12724-12732), and a peak was observed at 254362.
  • Example 9-1 the antibody synthesized in Example 7-2 into which one molecule of the thiol group and the azide group were introduced, the Fab of Trastuzumab into which the maleimide group synthesized in Example 8-2-5, and the implementation A tri-specific antibody was obtained by using the Fab of Pembrolizumab into which a PEG12-DBCO group was introduced synthesized in Example 8-3-4.
  • Tri-specific antibody analysis was performed by ESI-TOFMS analysis according to a previous report (Anal. Chem., 2019, 91, 20, 12724-12732), and a peak was observed at 255090.
  • Example 9-3 use the antibody (T-1-N3) into which only one molecule of azide group synthesized in Example 6-4 was introduced and the PEG12-DBCO group synthesized in Example 8-1-4. Bi-specific antibodies were obtained. Bi-specific antibody analysis was performed by ESI-TOFMS analysis according to a previous report (Anal. Chem., 2019, 91, 20, 12724-12732), and a peak was observed at 201356.
  • Recombinant human HER2-Fc (R&D Systems), recombinant human EGFR-Fc (R&D Systems), and recombinant human PD-1-Fc (R&D Systems) are available using the Biotin Labeling Kit (Dojind Systems). Biotinylation is performed using Biotin CAPture Kit ( Cytiva) was used to immobilize on the sensor chip. FcRn was immobilized on a sensor chip SA (Cytiva) using biotinylated recombinant human FcRn (Immunitrack). HBS-EP+buffer (Cytiva) was used as a running buffer for evaluating the affinity for the antigen. For affinity evaluation for FcRn, HBS-EP+buffer adjusted to pH 6.0 was used as the running buffer during binding, and HBS-EP+buffer with pH 7.4 was used as the running buffer during dissociation.
  • each antibody to the antigen and FcRn was evaluated by adding each antibody at 5 concentrations and using the single cycle kinetics method.
  • the obtained sensorgram was fitted with a 1:1 binding model, and the binding constant was calculated.
  • dissociation of the antibody was confirmed by adding a pH 7.4 buffer.
  • Example 10 Affinity evaluation of multispecific antibody by surface plasmon resonance (SPR) method (10-1) Affinity evaluation of Bi-specific antibody (Cetuximab-Trastuzumab Fab) (M-3)
  • SPR surface plasmon resonance
  • Bi -SPECIFIC antibodies CETUXIMAB -TRASTUZUMAB FAB
  • BI -SPECIFIC antibody Trastuzumab -CETUXIMAB FAB
  • Example 11 Evaluation of binding of multispecific antibodies to antigens on cells by flow cytometry
  • Cells include HER2-positive SKBR-3 cells, EGFR-positive A-431 cells, and PD-1-positive cells CD3 activated human T cells were used.
  • the binding evaluation was performed using flow cytometry method.
  • cetuximab bi-specific antibody (Cetuximab-Trastuzumab Fab) (M-3), bi-specific antibody (Trastuzumab-Cetuximab Fab) (M-4), Tri-specific antibody fic antibody (M -2) Fluorescence was observed in more than 98% of the cells with each antibody.
  • trastuzumab binds only to SKBR3 cells, which are HER2-positive cells, and cetuximab only binds to A-431 cells, which are EGFR-positive cells, while bi-specific antibody (Cetuximab-Trastuzumab Fab) (M -3), bi-specific antibody (Trastuzumab-Cetuximab Fab) (M-4), and tri-specific antibody (M-2) were confirmed to be able to bind to both SKBR3 cells and A-431 cells.
  • bi-specific antibody Cetuximab-Trastuzumab Fab
  • M-4 bi-specific antibody
  • M-2 tri-specific antibody
  • Example 12 Expression of VHH antibody (CD3-VHH, V-1) into which azidophenylalanine (AzF) is site-specifically introduced (12-1) Expression of plasmid vector (pPK14e) carrying AzFN3 DNA cassette in pPK5 vector Construction C. It is known that azidophenylalanine (AzF) can be site-specifically introduced into a target protein in secretory production of a heterologous protein in S. glutamicum (WO2023/282315). AzFN3 DNA cassette (SEQ ID NO: 43) was obtained by total synthesis with reference to WO2023/282315.
  • the AzFN3 DNA cassette contains the azidophenylalanyl-tRNA synthetase (AzFRS) gene and the ApaI site downstream thereof, and further downstream thereof the F1 promoter and the UAG codon linked downstream thereof that can be translated into azidophenylalanine (AzF). It contains a suppressor tRNA (tRNA CTA ) gene, an rrnC terminator downstream thereof, a KpnI site at the 5'-end, and an XbaI site at the 3'-end.
  • AzFRS azidophenylalanyl-tRNA synthetase
  • AzFRS is a modified tyrosyl-tRNA synthetase derived from the archaeal bacterium Methanococcus jannaschii that has the mutations Y32T/E107N/D158P/I159L/L162Q and is modified to use azidophenylalanine as a substrate (J.AM.CHEM.SOC .2002, 124, 9026-9027).
  • the base sequence of the AzFRS gene and the amino acid sequence of AzFRS are shown in SEQ ID NO: 44 and SEQ ID NO: 45, respectively.
  • tRNA CTA is a modified tyrosyl tRNA derived from the archaeon Methanococcus jannaschii that has been modified to have an anticodon (CTA) corresponding to UAG (amber).
  • CTA anticodon
  • the base sequence of the tRNA CTA gene and the base sequence of tRNA (Tyr) encoded by the gene are shown in SEQ ID NO: 46 and SEQ ID NO: 47, respectively.
  • Azidophenylalanyl tRNA binds to the UAG codon in mRNA during the protein translation process, the UAG codon is translated as azidophenylalanine, and a protein with azidophenylalanine introduced at the UAG codon position is synthesized.
  • a vector pPK14e incorporating the AzFN3 DNA cassette was constructed by integrating the fully synthesized AzFN3 DNA cassette (SEQ ID NO: 43) into the KpnI-XbaI site of the pPK5 vector described in WO2016/171224 by an infusion reaction.
  • In-Fusion registered trademark
  • HD Cloning Kit Tekara Bio
  • the base sequence was determined using BigDye (registered trademark) Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and 3500xL Genetic Analyzer (Applied Biosystems).
  • the pPK14e vector can be used as a basic vector for the expression of AzF-transduced proteins. That is, by subcloning the expression cassette of the target protein using the ApaI site of pPK14e, the AzFN3 DNA cassette (azidophenylalanyl-tRNA synthetase (AzFRS) gene and a suppressor capable of translating UAG codon into azidophenylalanine (AzF)) Expression cassettes for tRNA (tRNA CTA gene) and AzF transfection protein can be simultaneously expressed in one plasmid.
  • AzFRS azidophenylalanyl-tRNA synthetase
  • AzF azidophenylalanine
  • each secretory expression plasmid of CD3-VHH-PA24H6-WT and CD3-VHH-PA24H6-AzF As a VHH antibody that introduces azidophenylalanine, the anti-CD3-VHH antibody described in WO2015/095412 (CD3- VHH) was used. In order to introduce azidophenylalanine without modifying the internal CD3-VHH sequence, a modified CD3-VHH with a PA24 linker and a 6xHis tag added to the C-terminus of CD3-VHH was designed (CD3-VHH-PA24H6-WT). .
  • CD3-VHH-PA24H6 was designed to introduce AzF into the PA24 linker (between positions 141 and 142) of CD3-VHH-PA24H6-WT (CD3-VHH-PA24H6-AzF).
  • the designed amino acid sequences of CD3-VHH-PA24H6-WT and CD3-VHH-PA24H6-AzF are shown in SEQ ID NOs: 48 and 49, respectively.
  • the base sequences encoding these polypeptides are C. It was designed taking into consideration the codon usage frequency of C. glutamicum. Furthermore, C. The following expression cassettes were designed to enable secretory expression by C. glutamicum.
  • CD3-VHH-PA24H6-WT is C.
  • the protein was secreted and expressed as a fusion protein (hereinafter referred to as "CspAss-CD3-VHH-PA24H6-WT") consisting of 25 amino acid residues of the CspA signal peptide derived from P. stationis and CD3-VHH-PA24H6-WT.
  • CspAss-CD3-VHH-PA24H6-WT consisting of 25 amino acid residues of the CspA signal peptide derived from P. stationis and CD3-VHH-PA24H6-WT.
  • the nucleotide sequence and amino acid sequence encoding the designed CspAss-CD3-VHH-PA24H6-WT are shown in SEQ ID NOs: 50 and 51, respectively.
  • CD3-VHH-PA24H6-AzF is a C.I.
  • the protein was secreted and expressed as a fusion protein (hereinafter referred to as "CspAss-CD3-VHH-PA24H6-Amber") of 25 amino acid residues of the signal peptide of CspA derived from C. stationis and CD3-VHH-PA24H6-AzF.
  • CspAss-CD3-VHH-PA24H6-Amber gene is created by inserting a TAG triplet corresponding to the UAG codon (amber), which is one of the stop codons, in the base sequence of the CspAss-CD3-VHH-PA24H6-WT gene.
  • the nucleotide sequence and amino acid sequence encoding the designed CspAss-CD3-VHH-PA24H6-Amber are shown in SEQ ID NOs: 52 and 53, respectively.
  • SEQ ID NO: 53 (F at position 167 is azidated) MKRMKSLAAALTVAGAMLAAPVATAQVQLQESGGGLVQAGGSLRLSCAASGRTFSNYHMGWFRQAPGKERELVAAISGSGGSTYYTDSVKGRFTISRNNAKNTMSLQMSNLKPEDTGVYYC TTPTEKGSSIDYWGQGTQVTVSSGRYPYDVPDYAAPAAPAPAAAPAFAPAPAAAPAAPAAPAPAPHHHHHHHHHHH
  • CD3-VHH-PA24H6 expression cassette By inserting the totally synthesized DNA fragment (CD3-VHH-PA24H6 expression cassette) into the ApaI site of the pPK14e vector constructed in Example (12-1), wild-type and mutant CD3-VHH-PA24H6 Secretory expression plasmids pPK14e_CspAss-CD3-VHH-PA24H6-WT and pPK14e_CspAss-CD3-VHH-PA24H6-Amber were constructed, respectively. As a result of nucleotide sequencing of the inserted fragments, it was confirmed that each of the designed CD3-VHH-PA24H6+AzFN3-carrying vectors had been constructed.
  • Each of the obtained transformants was cultured at 25° C. for 120 hours in an MMTG liquid medium containing 0.3 mM azidophenylalanine and 25 mg/L kanamycin. After the completion of the culture, 1.0 ⁇ L of the culture supernatant obtained by centrifuging each culture solution was subjected to reduced SDS-PAGE using NuPAGE (registered trademark) 12% Bis-Tris Gel (Thermo Fisher Scientific), and then Quick - Staining was performed with CBB (Wako).
  • CD3-VHH-PA24H6-AzF (V-1)
  • the centrifuged supernatant of the CD3-VHH-PA24H6-AzF-expressing strain culture obtained in Example (12-3) was purified by 0.2 ⁇ m Sterile filtration was performed using a PVDF membrane filter.
  • the expressed CD3-VHH-PA24H6-AzF was purified using an affinity column using a 6xHis-tag at the C-terminus.
  • the 6xHis-tagged protein was purified using Ni-NTA Spin Kin (QIAGEN), and the purification conditions were in accordance with the manufacturer's recommended protocol.
  • Example (4-3) a hydroxylamine solution was added according to Example (4-3), and the mixture was shaken at room temperature for 1 hour to obtain a thiol group-introduced antibody (D-2).
  • D-2 a thiol group-introduced antibody
  • HIC-HPLC analysis was performed and it was confirmed that one molecule of thiol group was introduced.
  • One molecule of DBCO group was obtained by following the method described in WO2022154127 using Cetuximab (D-2) into which one molecule of thiol group synthesized in Example 13-2 and the DBCO group reagent (R2) described in WO2022154127.
  • the introduced antibody (D-3) was obtained.
  • HIC-HPLC analysis was performed according to a previous report (Anal. Chem., 2019, 91, 20, 12724-12732), and it was confirmed that one molecule of DBCO group was bound to Cetuximab.
  • the azidophenylalanine-introduced VHH synthesized in Example Z was added to the acetate buffer (50 mM sodium acetate, pH 5.5) solution of Cetuximab (D-3) to which one molecule of DBCO group synthesized in Example (13-3) was bonded. (3 equivalents to antibody) was added and shaken for 20 hours.
  • the reaction solution was purified using AKTA pure 25 (manufactured by Cytiva) to obtain an antibody (D-4) to which one molecule of VHH was bound.
  • ESI-TOFMS analysis was performed according to a previous report (Anal. Chem., 2019, 91, 20, 12724-12732), and a peak was observed at 170492.
  • Recombinant human EGFR-Fc was biotinylated using the Biotin Labeling Kit (Dojindo) and immobilized on the sensor chip using the Biotin CAPture Kit (Cytiva).
  • CD3 was immobilized on the sensor chip using biotinylated recombinant human CD3 ⁇ / ⁇ -Fc (ACRO Biosystems) and Biotin CAPture Kit (Cytiva).
  • HBS-EP+buffer (Cytiva) was used as the running buffer for evaluating the affinity for the antigen.
  • Cetuximab (D-4) with one molecule of VHH (V-1) can bind to EGFR and CD3 without impairing the binding affinity of the respective binding molecules, antibody and VHH. It turned out to be a heavy specific antibody.
  • Antibody (E-1) having one molecule of azide group and two molecules of thiol group obtained in Example 14-1 and the known substance MC-Val-Cit-PABA-MMAE (Organic & Biomolecular Chemistry, 2016, 14 , 9501-9518) to obtain an antibody (E-2) into which two molecules of Val-Cit-PABA-MMAE were introduced.
  • HIC-HPLC analysis was performed according to a previous report (WO2019/240287A1), and it was confirmed that two molecules of Val-Cit-PABA-MMAE were introduced.
  • Example (B-3) was added to an acetate buffer (50 mM sodium acetate, pH 5.5) solution of DAR2 ADC (E-2) having one molecule of azide group synthesized in Example (14-2).
  • An acetate buffer (50 mM sodium acetate, pH 5.5) solution (3 equivalents relative to the antibody) of the synthesized Fab of Cetuximab having a DBCO group was added, and the mixture was shaken for 40 hours.
  • the reaction solution was purified using AKTA pure 25 (manufactured by Cytiva) to obtain ADC (E-3) to which one Fab molecule was bound.
  • ESI-TOFMS analysis was performed according to a previous report (Anal. Chem., 2019, 91, 20, 12724-12732), and a peak was observed at 203036.
  • Example 15 Design of affinity substances and C. glutamicum secretion and expression (15-1) Outline of design of affinity substance In the present invention, it is necessary to design an affinity substance that contains two or more sites in the molecule that can bind to antibodies. That is, the affinity substance needs to be designed such that A) two or more antibody-binding sites are connected via a linker for connecting the antibody-binding sites.
  • the affinity substance is a polypeptide and a linker is bonded to the amino group of K (lysine) of the polypeptide
  • K lysine
  • polypeptide affinity substances were designed as shown in (i) to (n) below.
  • the N-terminus of the CspB tag is Q
  • the first residue Q can be pyroglutamylated to protect the N-terminal amino group. That is, in addition to the rules A), B), and C) above, consideration of the rule D) below is effective in expressing an affinity polypeptide using Corynex (registered trademark). D) Adding three QET residues to the N-terminus to improve secretion efficiency with Corynex (registered trademark)
  • the affinity polypeptides include the above-mentioned QET-Fc3K-PA48G-Fc3R, QET-Fc3K-PAS56-Fc3R, QET-Fc3K-PAS60-Fc3R, QET-Fc3K-PAS64-Fc3R, QET-Fc3K-PAS68-Fc3R, and QET.
  • QET-Fc3K-PA48G-Fc3R is a C. glutamicum ATCC 13869 strain, the N-terminal 3 amino acid residues QET of the CspB mature protein derived from the same strain, and the Fc3K-PA48G-Fc3R fusion protein (hereinafter referred to as "CspBss-QET-Fc3KR-PA48G”). ”) was secreted and expressed.
  • the nucleotide sequence and amino acid sequence encoding the designed CspBss-QET-Fc3KR-PA48G are shown in SEQ ID NOs: 60 and 61, respectively.
  • QET-Fc3K-PAS56-Fc3R is a C. glutamicum ATCC 13869 strain, the N-terminal 3 amino acid residues QET of the CspB mature protein derived from the same strain, and the Fc3K-PAS56-Fc3R fusion protein (hereinafter referred to as "CspBss-QET-Fc3KR-PAS56"). ”) was secreted and expressed.
  • the nucleotide sequence and amino acid sequence encoding the designed CspBss-QET-Fc3KR-PAS56 are shown in SEQ ID NOs: 62 and 63, respectively.
  • QET-Fc3K-PAS60-Fc3R is a C. glutamicum ATCC 13869 strain, the N-terminal 3 amino acid residues QET of the CspB mature protein derived from the same strain, and the Fc3K-PAS60-Fc3R fusion protein (hereinafter referred to as "CspBss-QET-Fc3KR-PAS60"). ”) was secreted and expressed.
  • the nucleotide sequence and amino acid sequence encoding the designed CspBss-QET-Fc3KR-PAS60 are shown in SEQ ID NOs: 64 and 65, respectively.
  • QET-Fc3K-PAS64-Fc3R is a C. glutamicum ATCC 13869 strain, the N-terminal 3 amino acid residues QET of the CspB mature protein derived from the same strain, and the Fc3K-PAS64-Fc3R fusion protein (hereinafter referred to as "CspBss-QET-Fc3KR-PAS64"). ”) was secreted and expressed.
  • the nucleotide sequence and amino acid sequence encoding the designed CspBss-QET-Fc3KR-PAS64 are shown in SEQ ID NOs: 66 and 67, respectively.
  • QET-Fc3K-PAS68-Fc3R is a C. glutamicum ATCC13869 strain, the N-terminal 3 amino acid residues QET of the CspB mature protein derived from the same strain, and the Fc3K-PAS68-Fc3R fusion protein (hereinafter referred to as "CspBss-QET-Fc3KR-PAS68"). ”) was secreted and expressed.
  • the nucleotide sequence and amino acid sequence encoding the designed CspBss-QET-Fc3KR-PAS68 are shown in SEQ ID NOs: 68 and 69, respectively.
  • QET-Fc3K-PAS80-Fc3R is C. glutamicum ATCC 13869 strain, the N-terminal 3 amino acid residues QET of the CspB mature protein derived from the same strain, and the Fc3K-PAS80-Fc3R fusion protein (hereinafter referred to as "CspBss-QET-Fc3KR-PAS80"). ”) was secreted and expressed.
  • the nucleotide sequence and amino acid sequence encoding the designed CspBss-QET-Fc3KR-PAS80 are shown in SEQ ID NOs: 70 and 71, respectively.
  • the YDK0107 ⁇ pmt1 strain constructed as described below was transformed using each plasmid, and YDK0107 ⁇ pmt1/pPK4_CspBss-QET-Fc3KR-PA48G strain, YDK0107 ⁇ pmt1/pPK4_CspBss-QET-Fc3KR-PAS56 strain, and YDK0107 ⁇ pmt1/pPK4_CspBss-QET-Fc3KR-PAS56 strain were transformed.
  • DK0107 ⁇ pmt1/pPK4_CspBss -QET-Fc3KR-PAS60 strain YDK0107 ⁇ pmt1/pPK4_CspBss-QET-Fc3KR-PAS64 strain, YDK0107 ⁇ pmt1/pPK4_CspBss-QET-Fc3KR-PAS68 strain, and YDK0107 ⁇ pmt1/pPK4_ CspBss-QET-Fc3KR-PAS strain 80 was obtained.
  • Each of the obtained transformants was cultured in an MMTG liquid medium containing 25 mg/L of kanamycin at 30°C for 72 hours.
  • O-mannosyltransferase PMT1 deficient protein O-mannosyltransferase PMT1 ( Martina Mahne et al., The Corynebacterium glutamicum gene pmt encoding a glycosyltransferase related to eukaryotic protei n-O-mannosyltransferases is essential for glycosylation of the resuscitation promoting factor (Rpf2) and other secreted prot eins. FEMS Microbiol Lett. 2006 Jun; 259 (2) :226-33.). Also, C.
  • C. glutamicum YDK0107 strain pBS5T ⁇ pmt1 constructed in (1) was used to construct C. glutamicum YDK0107 strain described in WO2016/171224.
  • glutamicum YDK0107 strain was transformed.
  • C. glutamicum YDK0107 strain is C. glutamicum YDK0107 strain. This is a natural mutant strain in which the W302C mutation was introduced into the phoS gene, which was obtained using the YDK010 strain of YDK010 as the parent strain (WO2016/171224).
  • C. glutamicum YDK010 strain is C. glutamicum YDK010 strain.
  • Example 16 (16-1) Synthesis of affinity reagent According to Example 3, the peptides (SEQ ID NOs: 54 to 59) synthesized in Example 15 were amidated to produce affinity substances (W-1, 2, 3, 4, 5 , 6) was prepared.
  • 1,1′-Carbodiimidazole (3.3 mg) and triethylamine (8.5 ⁇ L) were added to a DMF solution (1 mL) of commercially available Propargyl-PEG24-amine (BroadPharma, 51.7 mg), and the mixture was stirred at room temperature for 3 hours. After the reaction was completed, elution was performed by reverse phase preparative chromatography. Fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and then freeze-dried to obtain the modified reagent (PL-1) (34.7 mg).
  • PL-3 (1.5 mg) synthesized in (17-3) and PL-2 (1.1 mg) synthesized in (17-2) were dissolved in water (1.0 mL), and copper sulfate pentahydrate was dissolved. (0.33 ⁇ mol), L(+)-sodium ascorbate (0.83 ⁇ mol), and Tris(3-hydroxypropyltriazolylmethyl)amine (0.33 ⁇ mol) were added, and the mixture was stirred at room temperature for 20 minutes. After the reaction was completed, elution was performed by reverse phase preparative chromatography. Fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and then freeze-dried to obtain the above modified reagent (PL-4) (2.0 mg).
  • an affinity reagent (PL-5) was prepared by amidating PL-4 prepared in (17-4).
  • Example 18 Modification of Trastuzumab using affinity reagent (PL-5) (18-1) Modification of Trastuzumab using affinity reagent (PL-5)
  • Anti-human HER2 monoclonal antibody according to Example (4-1) Trastuzumab (Chugai Pharmaceutical Co., Ltd.) in acetate buffer (50mM Sodium acetate, pH 6.5) was added with a DMF solution (5 equivalent to the antibody) of the affinity reagent (PL-5) synthesized in Example (17-5). amount) and shaken for 1 hour.
  • Trastuzumab with one molecule of affinity reagent bound was obtained by purifying the reaction solution using Nap25 (manufactured by Cytiva).
  • ESI-TOFMS analysis was performed according to a previous report (Anal. Chem., 2019, 91, 20, 12724-12732), and it was confirmed that one molecule of the affinity reagent bound to Trastuzumab.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • Microbiology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Epidemiology (AREA)
  • Mycology (AREA)
  • Immunology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Cell Biology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne une technologie de modification chimique facile d'une seule chaîne lourde dans un motif structural d'anticorps (unité d'immunoglobuline comprenant deux chaînes lourdes et éventuellement deux chaînes légères). Plus particulièrement, la présente invention concerne un composé qui contient (A) une substance d'affinité contenant des première et seconde parties d'affinité qui ont une affinité pour la région constante dans la chaîne lourde d'un anticorps et (B) un groupe réactif avec l'anticorps, ou un sel dudit composé.
PCT/JP2023/020712 2022-06-02 2023-06-02 Substance d'affinité, composé, anticorps et leurs sels WO2023234416A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
JP2022090443 2022-06-02
JP2022-090443 2022-06-02
JP2022141780 2022-09-06
JP2022-141780 2022-09-06

Publications (1)

Publication Number Publication Date
WO2023234416A1 true WO2023234416A1 (fr) 2023-12-07

Family

ID=89025083

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2023/020712 WO2023234416A1 (fr) 2022-06-02 2023-06-02 Substance d'affinité, composé, anticorps et leurs sels

Country Status (1)

Country Link
WO (1) WO2023234416A1 (fr)

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018199337A1 (fr) * 2017-04-28 2018-11-01 味の素株式会社 Composé renfermant une substance ayant une affinité pour une protéine soluble, fraction clivable, et groupe réactif, ou sel de celui-ci
WO2019240288A1 (fr) * 2018-06-14 2019-12-19 味の素株式会社 Substance ayant une affinité pour un anticorps, et composé ou sel de celui-ci possédant un groupe fonctionnel bioorthogonal
WO2019240287A1 (fr) * 2018-06-14 2019-12-19 味の素株式会社 Composé comprenant une substance ayant une affinité pour un anticorps, site de clivage et groupe réactif, ou sel correspondant
WO2020090979A1 (fr) * 2018-10-31 2020-05-07 味の素株式会社 Composé comprenant une substance ayant une affinité pour un anticorps, site de clivage et groupe réactif ou sel correspondant
WO2021080008A1 (fr) * 2019-10-24 2021-04-29 国立大学法人 鹿児島大学 Procédé de fabrication de produit ccap monovalent
WO2021112249A1 (fr) * 2019-12-06 2021-06-10 味の素株式会社 Procédé de production d'un peptide ayant une activité physiologique, et peptide comprenant un lieur court

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018199337A1 (fr) * 2017-04-28 2018-11-01 味の素株式会社 Composé renfermant une substance ayant une affinité pour une protéine soluble, fraction clivable, et groupe réactif, ou sel de celui-ci
WO2019240288A1 (fr) * 2018-06-14 2019-12-19 味の素株式会社 Substance ayant une affinité pour un anticorps, et composé ou sel de celui-ci possédant un groupe fonctionnel bioorthogonal
WO2019240287A1 (fr) * 2018-06-14 2019-12-19 味の素株式会社 Composé comprenant une substance ayant une affinité pour un anticorps, site de clivage et groupe réactif, ou sel correspondant
WO2020090979A1 (fr) * 2018-10-31 2020-05-07 味の素株式会社 Composé comprenant une substance ayant une affinité pour un anticorps, site de clivage et groupe réactif ou sel correspondant
WO2021080008A1 (fr) * 2019-10-24 2021-04-29 国立大学法人 鹿児島大学 Procédé de fabrication de produit ccap monovalent
WO2021112249A1 (fr) * 2019-12-06 2021-06-10 味の素株式会社 Procédé de production d'un peptide ayant une activité physiologique, et peptide comprenant un lieur court

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
AUVERT G., PAULEAU Y., TONNEAU D.: "KINETICS OF LASER-ASSISTED CHEMICAL VAPOR DEPOSITION OF TUNGSTEN MICROSTRUCTURES.", JAPANESE JOURNAL OF APPLIED PHYSICS, JAPAN SOCIETY OF APPLIED PHYSICS, JP, vol. 31., no. 01., 1 January 1992 (1992-01-01), JP , pages 100 - 104., XP000955107, ISSN: 0021-4922, DOI: 10.1143/JJAP.31.100 *
HAYASHI YOSHIO, MUGURUMA, KYOHEI: "Basic research on antibody-dependent drug delivery technology using antibody-binding peptides,", DRUG DELIVERY SYSTEM., NIHON D D S GAKKAI, JAPAN, vol. 35, no. 3, 25 July 2020 (2020-07-25), Japan , pages 191 - 199, XP055921158, ISSN: 0913-5006, DOI: 10.2745/dds.35.191 *
ITO YUJI, ABDUR RAFIQUE: "Site-specific modification technology CCAP method that enables improvement of antibody functions and creation of highly functional antibody drugs", PHARMACIA, vol. 57, no. 7, 1 January 2021 (2021-01-01), pages 625 - 629, XP093116991 *
MATSUDA YUTAKA, TATSUYA OKUZUMI: "AJICAP: Expanding the therapeutic index by creating regioselective antibody-drug conjugates", PHARMACIA, vol. 57, no. 7, 1 January 2021 (2021-01-01), pages 646 - 650, XP093116981 *
YUTAKA MATSUDA, VERONICA ROBLES, MARIA-CHRISTINA MALINAO, JAMES SONG, BRIAN A. MENDELSOHN: "Comparison of Analytical Methods for Antibody–Drug Conjugates Produced by Chemical Site-Specific Conjugation: First-Generation AJICAP", ANALYTICAL CHEMISTRY, AMERICAN CHEMICAL SOCIETY, US, vol. 91, no. 20, 15 October 2019 (2019-10-15), US , pages 12724 - 12732, XP055767962, ISSN: 0003-2700, DOI: 10.1021/acs.analchem.9b02192 *

Similar Documents

Publication Publication Date Title
US20210300972A1 (en) Compound having affinity substance to antibody, cleavable portion, and reactive group, or salt thereof
US20210139541A1 (en) Compound having affinity substance to antibody and bioorthogonal functional group, or salt thereof
JP7413999B2 (ja) 抗体に対する親和性物質、切断性部分および反応性基を有する化合物またはその塩
JP7222347B2 (ja) 可溶性タンパク質に対する親和性物質、切断性部分および反応性基を有する化合物またはその塩
WO2023234416A1 (fr) Substance d'affinité, composé, anticorps et leurs sels
EP3774880A1 (fr) Réduction sélective d'anticorps
WO2023054706A1 (fr) Conjugué d'anticorps et de substance fonctionnelle ou de son sel, dérivé d'anticorps et composé ou ses sels à utiliser dans la production d'un conjugué ou de son sel
WO2022191283A1 (fr) Composé ou son sel, et anticorps ainsi obtenu
WO2023054714A1 (fr) Conjugué régiosélectif de substance fonctionnelle et d'anticorps ou sel dudit conjugué, dérivé d'anticorps et composé destinés à être utilisés dans la production dudit conjugué, ou sel dudit dérivé d'anticorps et de composé
US20240000965A1 (en) Compound or salt thereof, and antibody obtained by using the same
EP4279502A1 (fr) Composé ou son sel, et anticorps ainsi obtenu
JP2024010532A (ja) 化合物またはその塩
KR20240073034A (ko) 항체 및 기능성 물질의 위치 선택적인 컨쥬게이트 또는 그 염, 및 그 제조에 사용되는 항체 유도체 및 화합물 또는 그것들의 염
KR20240073035A (ko) 항체 및 기능성 물질의 컨쥬게이트 또는 그 염, 및 그 제조에 사용되는 항체 유도체 및 화합물 또는 그 염
CN118119639A (zh) 抗体和功能性物质的缀合物或其盐、以及其制造中使用的抗体衍生物和化合物或它们的盐

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23816173

Country of ref document: EP

Kind code of ref document: A1